AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM).METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12...AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM).METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors,yielding an HCEP cell line which was its growth performance,chromosome morphology,tumorigenicity and expression of marker proteins analyzed.In addition,the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light,electron and slit-lamp microscopies.RESULTS: HCEP cells proliferated to confluence in 3 weeks,which have been subcultured to passage 160.A continuous untransfected HCEP cell line,designated as utHCEPC01,was established with a population doubling time of 45.42 hours as was determined at passage 100.The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3.They,with no tumorigenicity,formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture,maintained expression of marker proteins including keratin 3 and integrin β1 and attached tightly to dAMs.The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original.CONCLUSION: An untransfected and non-tumorigenic HCEP cell line was established in this study.The cells maintained expression of marker proteins.The cell line was biocompatible with dAM.It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP,promising for the treatment of diseases caused by corneal epithelial disorders.展开更多
AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1(dectin-1) in human corneal epithelial(HCE) cells infected by fungus. · METHODS: A total of 20 cases of healthy donor corneas were group...AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1(dectin-1) in human corneal epithelial(HCE) cells infected by fungus. · METHODS: A total of 20 cases of healthy donor corneas were group A,and 20 patients(20 eyes) suffered from fungal keratitis(FK) composed group B. Real-time qPCR and immunohistochemistry were applied to detect dectin-1 expression in corneal epithelium of both groups. HCE cells were cultured with aspergillus fumigatus(AF) antigens in vitro. The expression of dectin-1 mRNA was measured by real-time qPCR at the stimulation of 0,4,8 and 24h separately. Dectin-1 protein was detected by immunocytochemistry at 0 and 24h separately. ·RESULTS: Dectin-1 expressed in corneal epithelium of normal persons and FK patients. Vitro cellular experiment showed that the expression of dectin-1 mRNA in HCE cells began to increase after stimulation of AF antigens at 4h,and dectin-1 protein expression increased after stimulation at 24h. · CONCLUSION: Dectin-1 expressed in corneal epithelium of normal persons. AF antigens stimulation can elevate the expression of dectin-1 in HCE cells in vitro.展开更多
AIM:To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium(TE-HCEP) with seeder cells from an untransfected HCEP cell line.· METHODS:The TE-HCEPs were rec...AIM:To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium(TE-HCEP) with seeder cells from an untransfected HCEP cell line.· METHODS:The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line,and scaffold carriers of denuded amniotic membrane(dAM) in air-liquid interface culture for 3,5,7 and 9 days,respectively.The specimens were examined with hematoxylin-eosin(HE) staining of paraffin-section,immunocytochemical staining,scanning and transmission electron microscopy.· RESULTS:During in vitro reconstruction of TE-HCEP,HCEP cells formed a 3-4,6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3,5 and 7 days,respectively.But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9.And the cells maintained positive expression of marker proteins(keratin 3 and keratin 12),cell-junction proteins(zonula occludens-1,E-cadherin,connexin 43 and integrin β1) and membrane transport protein of Na+-K+ ATPase.The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5.· CONCLUSION:The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo.The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells,including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation.The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.展开更多
BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an im...BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an important role in inflammation and GC progression.Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities.However,the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIM To assess whether tumor necrosis factor-α(TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODS The normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the antiinflammatory effects of quercetin.The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line.Cell migration was measured using Transwell assay.The expression of proto-oncogene tyrosine-protein kinase Src(cSrc),phospho(p)-c-Src,extracellular-signal-regulated kinase 2(ERK2),p-ERK1/2,c-Fos,p-c-Fos,nuclear factor kappa B(NF-κB/p65),and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity.p65 expression was detected by immunofluorescence.MMP-9 m RNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction(q RT–PCR)and gelatin zymography,respectively.RESULTS q RT-PCR and gelatin zymography showed that TNF-αinduced MMP-9 m RNA and protein expression in a dose-and time-dependent manner.These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-αantagonist(TNFR inhibitor)in a dose-and timedependent manner.Quercetin and TNF-αantagonists decreased the TNF-α-induced phosphorylation of c-Src,ERK1/2,c-Fos,and p65 in a dose-and time-dependent manner.Quercetin,TNF-αantagonist,PP1,U0126,and tanshinone IIA(TSIIA)reduced TNF-α-induced c-Fos phosphorylation and AP-1–Luciferase(Luc)activity in a dose-and time-dependent manner.Pretreatment with quercetin,TNF-αantagonist,PP1,U0126,or Bay 11-7082 reduced TNF-α-induced p65 phosphorylation and translocation and p65–Luc activity in a dose-and timedependent manner.TNF-αsignificantly increased GES-1 cell migration,and these results were reduced by pretreatment with quercetin or a TNF-αantagonist.CONCLUSION Quercetin significantly downregulates TNF-α-induced MMP-9 expression in GES-1 cells via the TNFR-c-Src–ERK1/2 and c-Fos or NF-κB pathways.展开更多
AIM: To discuss the effects of different concentrations of tetramethylpyrazine(TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell(SDHCEC) induced by hydrogen peroxide.METHOD...AIM: To discuss the effects of different concentrations of tetramethylpyrazine(TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell(SDHCEC) induced by hydrogen peroxide.METHODS: We detected the combined effects of TMP with concentrations ranging from 4 mg/m L to 0.03 mg/m L and 800 μM hydrogen peroxide on SDHCEC. The methyl thiazolyl tetrazolium(MTT) assay was processed at 3, 6and 12 h separately while the detection of cell apoptosis at 6h only by flow cytometry.RESULTS: The viability of SDHCEC with 0.5 mg/m L,0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L TMP joint with800 μM hydrogen peroxide at 3h and 6h was significantly higher than that with 800 μM hydrogen peroxide only, P <0.05. However, except 0.25 mg/m L, TMP with other concentrations joint with 800 μM hydrogen peroxide at12 h could not significantly inhibit decreased SDHCEC viability induced by 800 μM hydrogen peroxide. At 12 h,TMP of 0.5 mg/m L, 0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L could significantly inhibit SDHCEC early apoptosis induced by 800 μM hydrogen peroxide, most remarkable at 0.25 mg/m L TMP, P <0.05.CONCLUSION: Our results suggested that hydrogen peroxide can induce apoptosis related damage to SDHCEC. TMP can protect SDHCEC from the damage,and the protective effects may be associated with its anti-apoptosis mechanism.展开更多
AIM: To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1(Raf-1) and its role in the innate immune response of human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus.ME...AIM: To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1(Raf-1) and its role in the innate immune response of human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus.METHODS: HCECs were cultured in vitro.They were randomly divided into 4 groups,including control group,Aspergillus fumigatus group,GW5074(an inhibitor of Raf-1) group and Laminarin [an inhibitor of Dendriti-cell-associated C-type lectin 1(Dectin-1)] group.The protein expression level of total Raf-1 and p-Raf-1 was measured by Western blot.The expression of IL-6 and IL-8 m RNA in each group was detected by real-time polymerase chain reaction.RESULTS: In Aspergillus fumigatus group,total Raf-1 protein levels in HCECs remained unchanged at 5,15,30 and 45min after infection,while p-Raf-1 expression was significantly enhanced at 30 min after infection compared with control group.However,the expression of p-Raf-1 was apparently declined after treated with GW5074 or Laminarin compared with Aspergillus fumigatus group.The expression levels of IL-6,IL-8 m RNA were significantly increased after stimulation with fumigatus compared with control group.Pre-treated with GW5074 significantly inhibited Aspergillus fumigatus-induced upregulation of IL-8 and IL-6.CONCLUSION: Aspergillus fumigatus stimulation can elevate the expression of p-Raf-1 in HCECs in vitro.Dectin-1/Raf-1 signal pathway may play a role on regulating the expression of inflammatory cytokines,including IL-6 and IL-8.展开更多
AIM:To explore the feasibility that human amniotic epithelial cells(hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized hum...AIM:To explore the feasibility that human amniotic epithelial cells(hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells(S-ihCECs).METHODS:hAECs were isolated by enzyme digestion,and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA-DR.Recovered and cultured S-ihCECs,immunocytochemistry was used to detect the expression of CK3/12.The proliferation of S ihCECs handled by different concentrations of mitomycin was detected by CCK-8.The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK-8.After filtered out the optimal conditions,we collected S-ihCECs culture media for 5 days,then prepared conditioned medium to incubate hAECs,inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs.Quantitative real-time reverse transcription-polymerase chain reaction(QRT PCR) was carried out to evaluate the expression of Oct4,NANOG,PAX6,and CK12 in the differentiation period.Immunocytochemistry and western bloting were used to detect the expression of CK3/12.RESULTS:The culture media collected every 12h,from 20μg/mL mitomycin pretreatment S-ihCECs could significantly promote the proliferation of hAECs.In the period of differentiation,the morphology of differentiated hAECs was obviously different compared with the control group,and the distinctive CK3/12 for corneal epithelial cells was detected.CONCLUSION:This study showed that hAECs can differentiate into corneal epithelial-like cells by in vitro replication of the corneal epithelial microenvironment,using the culture media collected from S-ihCECs,and it is possible that S-ihCECs culture media could be used in corneal tissue engineering.展开更多
AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs wer...AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.展开更多
The damage of human corneal cells encounter with the problem of availability of corneal cells for replacement. Limitation of the source of corneal cells has been realized. An attempt of development of corneal epitheli...The damage of human corneal cells encounter with the problem of availability of corneal cells for replacement. Limitation of the source of corneal cells has been realized. An attempt of development of corneal epithelial-like cells from the human skin-derived precursor (hSKPs) has been made in this study. Combination of three essential growth factors: epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) could demonstrate successfully induction of hSKPs to differentiation into corneal cells. The induced cells expressed the appearance of markers of corneal epithelial cells as shown by the presence of keratin 3 (K3) by antibody label and Western blot assay. The K3 gene expression of induced hSKPs cells as shown by reverse transcription-polymerase chain reaction (RT-PCR) technology was also demonstrated. The presence of these markers at both gene and protein levels could lead to our conclusion that the directional transdifferentiation of hSKPs cells into corneal epithelial cells was successfully done under this cell induction protocol. The finding shows a newly available stem cell source can be obtained from easily available skin. Cells from autologous human skin might be used for corneal disorder treatment in future clinical application.展开更多
AIM: To detect the expression of transforming growth factor beta-induced gene (TGFBI) protein in human corneal tissue and overexpress it in the human corneal epithelial cells in order to discuss the function of TGFBI ...AIM: To detect the expression of transforming growth factor beta-induced gene (TGFBI) protein in human corneal tissue and overexpress it in the human corneal epithelial cells in order to discuss the function of TGFBI in the pathogenesis of corneal dystrophy. METHODS: Immunohistochemistry(IHC) was used to detect the expression of TGFBI in the human cornea tissue. TGFBI cDNA was obtained by reverse transcription-PCR from human corneal total RNA extracted from cornea transplant donor and cloned into pCMV-N-HA vector. The recombinant pCMV-N-HA-TGFBI plasmid transfected human corneal epithelial cells. Forty-eight hours later, mRNA and proteins were harvested from cells for real-time PCR analysis and western blot assay respectively.RESULTS: IHC indicated TGFBI mainly exist below the human corneal epithelium layer. Transfection of recombinant pCMV-N-HA-TGFBI into human corneal epithelial cells resulted in effective expression of TGFBI, as shown by increased mRNA level detected by real-time PCR as well as increased protein level detected by Western blot. Meanwhile the result of real-time PCR and Western blot shown the expression of MMP1,MMP3(matrix metalloproteinases MMP) increased while the expressin of TIMP1 (tissue inhibitors of matrix metalloproteinases TIMP) decreased. CONCLUSION: TGFBI mainly exists below the corneal epithelial layer, recombinant eukaryotic expression vector harboring human TGFBI cDNA was obtained and efficiently overexpressed in human corneal epithelial cells. Meanwhile the TGFBI overexpression in human corneal epithelial cells result in MMP1, MMP3 increasing and TIMP1 decreasing. The result might be helpful for studying the function and role of TGFBI in pathogenesis of corneal dystrophy.展开更多
AIM: To evaluate the early expression of mannose-binding lectin 2(MBL2) in human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus(AF).METHODS: HCECs cultured in vitro with AF antigens and sampled at 0...AIM: To evaluate the early expression of mannose-binding lectin 2(MBL2) in human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus(AF).METHODS: HCECs cultured in vitro with AF antigens and sampled at 0, 0.5, 1, 2, 4, 6 and 8h. The expression of MBL2 m RNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction(RT-PCR). The expression of MBL2 protein in supernatant fluid was shown by enzyme linked immunosorbent assay(ELISA). MBL2 protein in HCECs was detected by immunocytochemistry at 0 and 24 h.RESULTS: MBL2 m RNA and protein are expressed in normal HCECs. The expression of MBL2 m RNA and protein in supernatant fluid begin to increase after being stimulated with AF antigens. The most significantly peak of MBL2 m RNA is in 2h. The protein of MBL2 in supernatant fluid decrease gradually after 0.5h. The protein in HCECs expression increase after stimulation of24 h.· CONCLUSION: MBL2 receptor expressed in normal HCECs in vitro. The stimulation by AF antigens can increase the early expression of it.展开更多
AIM:To study the effect of renalase peptide,RP-220,on cell viability of human corneal epithelial cells after alkali insult.METHODS:A dose-response relationship between cell viability and exposure to NaOH solution were...AIM:To study the effect of renalase peptide,RP-220,on cell viability of human corneal epithelial cells after alkali insult.METHODS:A dose-response relationship between cell viability and exposure to NaOH solution were characterized using cultured human corneal epithelial cells.Viability of corneal epithelial cells was determined using commercially available MTT and CyQUANT?assays.RESULTS:At a concentration of 6 mmol/L,insult with NaOH leads to reduced corneal epithelial cell viability by approximately 30%.This reduced viability was prevented by treating the cells after initial insult with the 20-amino acid renalase derived peptide(RP-220).CONCLUSION:RP-220 has a pro-survival role for RP-220 following alkaline insult to corneal epithelial cells.展开更多
Objective:To investigate the regulation effect of pigment epithelium-derived factor(PEDF) on the growth of human lens endothelial cells(LECs) and related mechanisms in mm and in vim.Methods:In the part of in vivo stud...Objective:To investigate the regulation effect of pigment epithelium-derived factor(PEDF) on the growth of human lens endothelial cells(LECs) and related mechanisms in mm and in vim.Methods:In the part of in vivo study,82 eyes of 82 patients with age-related cataract were included to collect the central lens anterior capsule(diameter at 5.0 to 5.5 mm) with the informed consent of surgery for patients.The selected specimens were divided into the LECs low density group and high density group with 20 specimens for each group based on hematoxylin and eosin staining results.The relative expression level of PEDF mRNA in LECs was detected by reverse transcription PCR.In the part of in vitro study,LEC line(HLEB3) was cultured and 50 ng/mL PEDF was added in media for 72 h in PEDF culture group,while normally cultured cells were used as the control group.The percentage of LECs at G_0and S phases and apoptotic rate of cells were assayed by using flow cytometry with annexin V-FTTC/7-AAD double staining method.Intracellular expression of vascular endothelial growth factor(VEGF) mRNA was detected by real-time fluorescence quantitative PCR.Results:The central anterior subcapsular LECs density and relative expression level of PEDF mRNA were lower than those of high density group.There were no significant differences between two groups(P=0.168).The apoptotic rate in the PEDF culture group was significantly reduced in comparison with the control group(P<0.001).In addition,the expression level of VEGF mRNA was lower in the PEDF culture group compared with the control group(P<0.001).Conclusions:In human eyes,PEDF may function as cytotropic factor to promote survival of LECs through anti-apoptosis and reducing-expression of VEGF.Decrease of PEDF content in LECs probably modulates the pathophysiological process of lens cells and further cataractogenesis.展开更多
Background:The goal was to explore the protective effect and potential mechanism of amniotic membrane extracts(AME)on the ocular surface exposed to benzalkonium chloride(BAC).Methods:The human corneal epithelial cell(...Background:The goal was to explore the protective effect and potential mechanism of amniotic membrane extracts(AME)on the ocular surface exposed to benzalkonium chloride(BAC).Methods:The human corneal epithelial cell(HCEC)line SD-HCEC1s was cultured in 5 groups:normal control(NC),NC+AME,BAC,BAC+NC,and BAC+AME.Cell viability analysis,flow cytometry analysis,real-time polymerase chain reaction(PCR),and western blot were employed to measure changes in cell function.Matrix metalloproteinases(MMPs)and inflammatory cytokines were assayed by enzyme-linked immunosorbent assay(ELISA)and activity assays.Results:Real-time PCR and western blot analysis demonstrated that the expressional level of caspase-8 was increased while the levels of Muc1,Muc4,and Muc16 were decreased after treatment with 0.02%BAC for 1 h.When the SD-HCEC1s were withdrawn from the BAC and switched to media containing 10%AME for 2 days,the expression level of capsase-8 was decreased while the levels of Muc1,Muc4,and Muc16 were increased.Real-time PCR and ELISA demonstrated that the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,interleukin(IL)-1β,IL-6,and tumor necrosis factor-alpha(TNF-α)were significantly increased after treatment with 0.02%BAC,whereas those of MMP-8 were decreased.When the 0.02%BAC was withdrawn and the SD-HCEC1s were cultured in 10%AME,the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,IL-1β,IL-6,and TNF-αwere decreased,while those of MMP-8 were increased.MMP-8 activity assays confirmed that IL-1βand TNF-αdownregulated the protein levels of MMP-8.Conclusions:AME protects SD-HCEC1s when stressed in BAC via upregulation of MMP-8 and downregulation of IL-1βand TNF-α.AME may have the potential functions to be employed as a topical adjunctive therapy in eyes chronically exposed to BAC.展开更多
·AIM:To evaluate the biological functions of tissue- engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models.·METHODS:TE-HCEPs were recons...·AIM:To evaluate the biological functions of tissue- engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models.·METHODS:TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy.·RESULTS:After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575μm in thickness during the monitoring period. A 4-5 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. ·CONCLUSION:The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.展开更多
AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of...AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.展开更多
Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this dise...Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and-18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin(CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1,-7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition(EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.展开更多
Purpose: To investigate the cytotoxicity of soft contact lens multi-purpose care solutions which are now in common use in China.Methods: The cell culture method was used. Cytotoxicity was indicated by significant incr...Purpose: To investigate the cytotoxicity of soft contact lens multi-purpose care solutions which are now in common use in China.Methods: The cell culture method was used. Cytotoxicity was indicated by significant increases in the number of dead cells relative to controls.Results: Cells were exposed to soft contact lens care solutions for 15 min. They were irregular in shape and variable in size. The intercellular space increased and variable in size.The in-tercelluar space increased and the cells became scrunken. With the time of exposure elongated , damage of cells became more severe.Conclusions: Four kinds of soft contact lens multi-purpose care solutions may have harmful effects on the culture of human corneal epithelial cells. Soaked lenses should be rinsed with saline before being placed in the eyes in order to reduce the potential toxicity of contact lens care solutions. Eye science 1998; 14 :45 - 47.展开更多
In this paper, quaternary 8-(1-acylethene-1-yl)-13-methylcoptisine chlorides targeting thioredoxin reductases(TrxRs) were designed to test the growth inhibitory activity against human cancer cell lines and the effect ...In this paper, quaternary 8-(1-acylethene-1-yl)-13-methylcoptisine chlorides targeting thioredoxin reductases(TrxRs) were designed to test the growth inhibitory activity against human cancer cell lines and the effect on viability of the normal intestinal epithelial cell-6(IEC-6) in vitro and to evaluate structure-activity relationship(SAR). The introduced α, β-unsaturated ketone groups at C-8 consisting of n-alkanoyls possessing five to ten carbons or aroyls or cyclohexylcarbonyl increased the tested activity against the target cancer cell lines. By and large, this type of improvement was increasingly graced by the elongation of the aliphatic chain of the n-alkanoyls in the range of less than ten carbon atoms. The relatively more polar 1-acylethene-1-yls displayed no effect on improving the activity. All the explored aroyls showed significant effect on improving the activity of the target compounds against the tested cancer cell lines with no SAR being observed. The findings of this study suggested that oil/water partition coefficient of the test compounds was one of the key factors impacting the target activity against the tested cancer cell lines. At the concentration of 10 μmol/L, except for the compounds with n-alkanoyls possessing seven or more carbons or with α-naphthoyl, none of the other compounds displayed obvious cytotoxicity on normal IEC-6 cell when co-incubated. The survival rate of IEC-6 cell ranged from 75% to 100% for the noncytotoxic compounds.展开更多
基金Supported by National High Technology Research and Development Program ("863" Program) of China(No. 2006AA02A132)
文摘AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM).METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors,yielding an HCEP cell line which was its growth performance,chromosome morphology,tumorigenicity and expression of marker proteins analyzed.In addition,the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light,electron and slit-lamp microscopies.RESULTS: HCEP cells proliferated to confluence in 3 weeks,which have been subcultured to passage 160.A continuous untransfected HCEP cell line,designated as utHCEPC01,was established with a population doubling time of 45.42 hours as was determined at passage 100.The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3.They,with no tumorigenicity,formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture,maintained expression of marker proteins including keratin 3 and integrin β1 and attached tightly to dAMs.The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original.CONCLUSION: An untransfected and non-tumorigenic HCEP cell line was established in this study.The cells maintained expression of marker proteins.The cell line was biocompatible with dAM.It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP,promising for the treatment of diseases caused by corneal epithelial disorders.
基金Supported by National Natural Science Foundation of China(No.81170825No.81300730)
文摘AIM: To evaluate the expression of dendritic cell-associated C-type lectin-1(dectin-1) in human corneal epithelial(HCE) cells infected by fungus. · METHODS: A total of 20 cases of healthy donor corneas were group A,and 20 patients(20 eyes) suffered from fungal keratitis(FK) composed group B. Real-time qPCR and immunohistochemistry were applied to detect dectin-1 expression in corneal epithelium of both groups. HCE cells were cultured with aspergillus fumigatus(AF) antigens in vitro. The expression of dectin-1 mRNA was measured by real-time qPCR at the stimulation of 0,4,8 and 24h separately. Dectin-1 protein was detected by immunocytochemistry at 0 and 24h separately. ·RESULTS: Dectin-1 expressed in corneal epithelium of normal persons and FK patients. Vitro cellular experiment showed that the expression of dectin-1 mRNA in HCE cells began to increase after stimulation of AF antigens at 4h,and dectin-1 protein expression increased after stimulation at 24h. · CONCLUSION: Dectin-1 expressed in corneal epithelium of normal persons. AF antigens stimulation can elevate the expression of dectin-1 in HCE cells in vitro.
基金Supported by National High Technology Research and Development Program("863" Program) of China(No.2006AA02A132)
文摘AIM:To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium(TE-HCEP) with seeder cells from an untransfected HCEP cell line.· METHODS:The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line,and scaffold carriers of denuded amniotic membrane(dAM) in air-liquid interface culture for 3,5,7 and 9 days,respectively.The specimens were examined with hematoxylin-eosin(HE) staining of paraffin-section,immunocytochemical staining,scanning and transmission electron microscopy.· RESULTS:During in vitro reconstruction of TE-HCEP,HCEP cells formed a 3-4,6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3,5 and 7 days,respectively.But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9.And the cells maintained positive expression of marker proteins(keratin 3 and keratin 12),cell-junction proteins(zonula occludens-1,E-cadherin,connexin 43 and integrin β1) and membrane transport protein of Na+-K+ ATPase.The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5.· CONCLUSION:The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo.The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells,including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation.The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.
基金Ministry of Science and Technology,Taiwan,No.MOST 108-2320-B-255-002-MY3 and No.MOST 110-2635-B-255-001Chang Gung Medical Research Foundation,Taoyuan,Taiwan,No.CMRPF1I0031,No.CMRPF1L0081,No.CMRPF1L0021,No.CMRPF1L0041,and No.CMRPF1I0042Chang Gung University of Science and Technology,Taoyuan,Taiwan,No.ZRRPF3K0111 and No.ZRRPF3L0091。
文摘BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an important role in inflammation and GC progression.Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities.However,the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIM To assess whether tumor necrosis factor-α(TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODS The normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the antiinflammatory effects of quercetin.The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line.Cell migration was measured using Transwell assay.The expression of proto-oncogene tyrosine-protein kinase Src(cSrc),phospho(p)-c-Src,extracellular-signal-regulated kinase 2(ERK2),p-ERK1/2,c-Fos,p-c-Fos,nuclear factor kappa B(NF-κB/p65),and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity.p65 expression was detected by immunofluorescence.MMP-9 m RNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction(q RT–PCR)and gelatin zymography,respectively.RESULTS q RT-PCR and gelatin zymography showed that TNF-αinduced MMP-9 m RNA and protein expression in a dose-and time-dependent manner.These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-αantagonist(TNFR inhibitor)in a dose-and timedependent manner.Quercetin and TNF-αantagonists decreased the TNF-α-induced phosphorylation of c-Src,ERK1/2,c-Fos,and p65 in a dose-and time-dependent manner.Quercetin,TNF-αantagonist,PP1,U0126,and tanshinone IIA(TSIIA)reduced TNF-α-induced c-Fos phosphorylation and AP-1–Luciferase(Luc)activity in a dose-and time-dependent manner.Pretreatment with quercetin,TNF-αantagonist,PP1,U0126,or Bay 11-7082 reduced TNF-α-induced p65 phosphorylation and translocation and p65–Luc activity in a dose-and timedependent manner.TNF-αsignificantly increased GES-1 cell migration,and these results were reduced by pretreatment with quercetin or a TNF-αantagonist.CONCLUSION Quercetin significantly downregulates TNF-α-induced MMP-9 expression in GES-1 cells via the TNFR-c-Src–ERK1/2 and c-Fos or NF-κB pathways.
基金Supported by Guangdong Administration of Traditional Chinese Medicine (No.2007095)
文摘AIM: To discuss the effects of different concentrations of tetramethylpyrazine(TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell(SDHCEC) induced by hydrogen peroxide.METHODS: We detected the combined effects of TMP with concentrations ranging from 4 mg/m L to 0.03 mg/m L and 800 μM hydrogen peroxide on SDHCEC. The methyl thiazolyl tetrazolium(MTT) assay was processed at 3, 6and 12 h separately while the detection of cell apoptosis at 6h only by flow cytometry.RESULTS: The viability of SDHCEC with 0.5 mg/m L,0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L TMP joint with800 μM hydrogen peroxide at 3h and 6h was significantly higher than that with 800 μM hydrogen peroxide only, P <0.05. However, except 0.25 mg/m L, TMP with other concentrations joint with 800 μM hydrogen peroxide at12 h could not significantly inhibit decreased SDHCEC viability induced by 800 μM hydrogen peroxide. At 12 h,TMP of 0.5 mg/m L, 0.25 mg/m L, 0.125 mg/m L and 0.06 mg/m L could significantly inhibit SDHCEC early apoptosis induced by 800 μM hydrogen peroxide, most remarkable at 0.25 mg/m L TMP, P <0.05.CONCLUSION: Our results suggested that hydrogen peroxide can induce apoptosis related damage to SDHCEC. TMP can protect SDHCEC from the damage,and the protective effects may be associated with its anti-apoptosis mechanism.
基金Supported by National Natural Science Foundation of China(No.81170825No.81470609+3 种基金No.81500695)Specialized Research Fund for the Doctoral Program of Higher Education(No.20123706110003)the Youth Natural Science Foundation of Shandong Province(No.ZR2013HQ007)the Key Project of Natural Science Foundation of Shandong Province(No.ZR2012HZ001)
文摘AIM: To investigate the expression of the v-raf-1murine leukemia viral oncogene homolog 1(Raf-1) and its role in the innate immune response of human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus.METHODS: HCECs were cultured in vitro.They were randomly divided into 4 groups,including control group,Aspergillus fumigatus group,GW5074(an inhibitor of Raf-1) group and Laminarin [an inhibitor of Dendriti-cell-associated C-type lectin 1(Dectin-1)] group.The protein expression level of total Raf-1 and p-Raf-1 was measured by Western blot.The expression of IL-6 and IL-8 m RNA in each group was detected by real-time polymerase chain reaction.RESULTS: In Aspergillus fumigatus group,total Raf-1 protein levels in HCECs remained unchanged at 5,15,30 and 45min after infection,while p-Raf-1 expression was significantly enhanced at 30 min after infection compared with control group.However,the expression of p-Raf-1 was apparently declined after treated with GW5074 or Laminarin compared with Aspergillus fumigatus group.The expression levels of IL-6,IL-8 m RNA were significantly increased after stimulation with fumigatus compared with control group.Pre-treated with GW5074 significantly inhibited Aspergillus fumigatus-induced upregulation of IL-8 and IL-6.CONCLUSION: Aspergillus fumigatus stimulation can elevate the expression of p-Raf-1 in HCECs in vitro.Dectin-1/Raf-1 signal pathway may play a role on regulating the expression of inflammatory cytokines,including IL-6 and IL-8.
基金National Natural Science Foundation of China(No.30872808)
文摘AIM:To explore the feasibility that human amniotic epithelial cells(hAECs) have the potential to differentiate into corneal epithelial-like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells(S-ihCECs).METHODS:hAECs were isolated by enzyme digestion,and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA-DR.Recovered and cultured S-ihCECs,immunocytochemistry was used to detect the expression of CK3/12.The proliferation of S ihCECs handled by different concentrations of mitomycin was detected by CCK-8.The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK-8.After filtered out the optimal conditions,we collected S-ihCECs culture media for 5 days,then prepared conditioned medium to incubate hAECs,inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs.Quantitative real-time reverse transcription-polymerase chain reaction(QRT PCR) was carried out to evaluate the expression of Oct4,NANOG,PAX6,and CK12 in the differentiation period.Immunocytochemistry and western bloting were used to detect the expression of CK3/12.RESULTS:The culture media collected every 12h,from 20μg/mL mitomycin pretreatment S-ihCECs could significantly promote the proliferation of hAECs.In the period of differentiation,the morphology of differentiated hAECs was obviously different compared with the control group,and the distinctive CK3/12 for corneal epithelial cells was detected.CONCLUSION:This study showed that hAECs can differentiate into corneal epithelial-like cells by in vitro replication of the corneal epithelial microenvironment,using the culture media collected from S-ihCECs,and it is possible that S-ihCECs culture media could be used in corneal tissue engineering.
基金Supported by Biomedical Research Institute Grant(No.2009-39)Pusan National University Hospital
文摘AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.
基金Supported by Stem Cell Project,National Research Council of Thailand (NRCT),Cell Engineering and Tissue Growth, Institute of Molecular Biosciences and Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Thailand
文摘The damage of human corneal cells encounter with the problem of availability of corneal cells for replacement. Limitation of the source of corneal cells has been realized. An attempt of development of corneal epithelial-like cells from the human skin-derived precursor (hSKPs) has been made in this study. Combination of three essential growth factors: epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) could demonstrate successfully induction of hSKPs to differentiation into corneal cells. The induced cells expressed the appearance of markers of corneal epithelial cells as shown by the presence of keratin 3 (K3) by antibody label and Western blot assay. The K3 gene expression of induced hSKPs cells as shown by reverse transcription-polymerase chain reaction (RT-PCR) technology was also demonstrated. The presence of these markers at both gene and protein levels could lead to our conclusion that the directional transdifferentiation of hSKPs cells into corneal epithelial cells was successfully done under this cell induction protocol. The finding shows a newly available stem cell source can be obtained from easily available skin. Cells from autologous human skin might be used for corneal disorder treatment in future clinical application.
基金Support by 2010 Guangdong Province Natural Science Fund(No.10151063201000044)2011 Guangdong Province Natural Science Fund,China(No.S2011010004186)2011 Central University Basic Research Special Fund and Jinan University Scientific Cultivation and Innovation Fund,China(No.21611446)
文摘AIM: To detect the expression of transforming growth factor beta-induced gene (TGFBI) protein in human corneal tissue and overexpress it in the human corneal epithelial cells in order to discuss the function of TGFBI in the pathogenesis of corneal dystrophy. METHODS: Immunohistochemistry(IHC) was used to detect the expression of TGFBI in the human cornea tissue. TGFBI cDNA was obtained by reverse transcription-PCR from human corneal total RNA extracted from cornea transplant donor and cloned into pCMV-N-HA vector. The recombinant pCMV-N-HA-TGFBI plasmid transfected human corneal epithelial cells. Forty-eight hours later, mRNA and proteins were harvested from cells for real-time PCR analysis and western blot assay respectively.RESULTS: IHC indicated TGFBI mainly exist below the human corneal epithelium layer. Transfection of recombinant pCMV-N-HA-TGFBI into human corneal epithelial cells resulted in effective expression of TGFBI, as shown by increased mRNA level detected by real-time PCR as well as increased protein level detected by Western blot. Meanwhile the result of real-time PCR and Western blot shown the expression of MMP1,MMP3(matrix metalloproteinases MMP) increased while the expressin of TIMP1 (tissue inhibitors of matrix metalloproteinases TIMP) decreased. CONCLUSION: TGFBI mainly exists below the corneal epithelial layer, recombinant eukaryotic expression vector harboring human TGFBI cDNA was obtained and efficiently overexpressed in human corneal epithelial cells. Meanwhile the TGFBI overexpression in human corneal epithelial cells result in MMP1, MMP3 increasing and TIMP1 decreasing. The result might be helpful for studying the function and role of TGFBI in pathogenesis of corneal dystrophy.
基金Supported by National Natural Science Foundation of China(No.81170825No.81300730)
文摘AIM: To evaluate the early expression of mannose-binding lectin 2(MBL2) in human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus(AF).METHODS: HCECs cultured in vitro with AF antigens and sampled at 0, 0.5, 1, 2, 4, 6 and 8h. The expression of MBL2 m RNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction(RT-PCR). The expression of MBL2 protein in supernatant fluid was shown by enzyme linked immunosorbent assay(ELISA). MBL2 protein in HCECs was detected by immunocytochemistry at 0 and 24 h.RESULTS: MBL2 m RNA and protein are expressed in normal HCECs. The expression of MBL2 m RNA and protein in supernatant fluid begin to increase after being stimulated with AF antigens. The most significantly peak of MBL2 m RNA is in 2h. The protein of MBL2 in supernatant fluid decrease gradually after 0.5h. The protein in HCECs expression increase after stimulation of24 h.· CONCLUSION: MBL2 receptor expressed in normal HCECs in vitro. The stimulation by AF antigens can increase the early expression of it.
基金Supported by the resources of the Central Texas Veterans Health Care System (Temple, TX)the Central Texas Veterans Health Care System Research Service
文摘AIM:To study the effect of renalase peptide,RP-220,on cell viability of human corneal epithelial cells after alkali insult.METHODS:A dose-response relationship between cell viability and exposure to NaOH solution were characterized using cultured human corneal epithelial cells.Viability of corneal epithelial cells was determined using commercially available MTT and CyQUANT?assays.RESULTS:At a concentration of 6 mmol/L,insult with NaOH leads to reduced corneal epithelial cell viability by approximately 30%.This reduced viability was prevented by treating the cells after initial insult with the 20-amino acid renalase derived peptide(RP-220).CONCLUSION:RP-220 has a pro-survival role for RP-220 following alkaline insult to corneal epithelial cells.
基金Supported by Scientific Rescarch Projects of Science and Technology Commission of Shanghai Municipality(No.11DZ1921208)
文摘Objective:To investigate the regulation effect of pigment epithelium-derived factor(PEDF) on the growth of human lens endothelial cells(LECs) and related mechanisms in mm and in vim.Methods:In the part of in vivo study,82 eyes of 82 patients with age-related cataract were included to collect the central lens anterior capsule(diameter at 5.0 to 5.5 mm) with the informed consent of surgery for patients.The selected specimens were divided into the LECs low density group and high density group with 20 specimens for each group based on hematoxylin and eosin staining results.The relative expression level of PEDF mRNA in LECs was detected by reverse transcription PCR.In the part of in vitro study,LEC line(HLEB3) was cultured and 50 ng/mL PEDF was added in media for 72 h in PEDF culture group,while normally cultured cells were used as the control group.The percentage of LECs at G_0and S phases and apoptotic rate of cells were assayed by using flow cytometry with annexin V-FTTC/7-AAD double staining method.Intracellular expression of vascular endothelial growth factor(VEGF) mRNA was detected by real-time fluorescence quantitative PCR.Results:The central anterior subcapsular LECs density and relative expression level of PEDF mRNA were lower than those of high density group.There were no significant differences between two groups(P=0.168).The apoptotic rate in the PEDF culture group was significantly reduced in comparison with the control group(P<0.001).In addition,the expression level of VEGF mRNA was lower in the PEDF culture group compared with the control group(P<0.001).Conclusions:In human eyes,PEDF may function as cytotropic factor to promote survival of LECs through anti-apoptosis and reducing-expression of VEGF.Decrease of PEDF content in LECs probably modulates the pathophysiological process of lens cells and further cataractogenesis.
基金This study was supported by the“Yangcheng Scholar”Youth Research Backbone Training Project of Guangzhou Municipal College(No.1201581612)Guangzhou Science and Technology Project(No.201804010038)Guangdong Natural Science Foundation of China(2020A1515010276,No.2015A030313479).
文摘Background:The goal was to explore the protective effect and potential mechanism of amniotic membrane extracts(AME)on the ocular surface exposed to benzalkonium chloride(BAC).Methods:The human corneal epithelial cell(HCEC)line SD-HCEC1s was cultured in 5 groups:normal control(NC),NC+AME,BAC,BAC+NC,and BAC+AME.Cell viability analysis,flow cytometry analysis,real-time polymerase chain reaction(PCR),and western blot were employed to measure changes in cell function.Matrix metalloproteinases(MMPs)and inflammatory cytokines were assayed by enzyme-linked immunosorbent assay(ELISA)and activity assays.Results:Real-time PCR and western blot analysis demonstrated that the expressional level of caspase-8 was increased while the levels of Muc1,Muc4,and Muc16 were decreased after treatment with 0.02%BAC for 1 h.When the SD-HCEC1s were withdrawn from the BAC and switched to media containing 10%AME for 2 days,the expression level of capsase-8 was decreased while the levels of Muc1,Muc4,and Muc16 were increased.Real-time PCR and ELISA demonstrated that the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,interleukin(IL)-1β,IL-6,and tumor necrosis factor-alpha(TNF-α)were significantly increased after treatment with 0.02%BAC,whereas those of MMP-8 were decreased.When the 0.02%BAC was withdrawn and the SD-HCEC1s were cultured in 10%AME,the mRNA and protein levels of MMP-1,MMP-3,MMP-13,CXCL1,IL-1β,IL-6,and TNF-αwere decreased,while those of MMP-8 were increased.MMP-8 activity assays confirmed that IL-1βand TNF-αdownregulated the protein levels of MMP-8.Conclusions:AME protects SD-HCEC1s when stressed in BAC via upregulation of MMP-8 and downregulation of IL-1βand TNF-α.AME may have the potential functions to be employed as a topical adjunctive therapy in eyes chronically exposed to BAC.
基金National High Technology Research and Development Program ("863"Program) of China (No.2006AA 02A132)
文摘·AIM:To evaluate the biological functions of tissue- engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models.·METHODS:TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy.·RESULTS:After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575μm in thickness during the monitoring period. A 4-5 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. ·CONCLUSION:The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.
基金Supported by National Natural Science Foundation of China(No.81273212,81100651)Project of Science and Technology of Shandong Province(No.2014GSF118044)
文摘AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.
基金Supported by Ministry of Education,Culture,Sports Science,and Technology,and the Ministry of Health,Labour and Welfare of Japan
文摘Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and-18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin(CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1,-7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition(EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.
文摘Purpose: To investigate the cytotoxicity of soft contact lens multi-purpose care solutions which are now in common use in China.Methods: The cell culture method was used. Cytotoxicity was indicated by significant increases in the number of dead cells relative to controls.Results: Cells were exposed to soft contact lens care solutions for 15 min. They were irregular in shape and variable in size. The intercellular space increased and variable in size.The in-tercelluar space increased and the cells became scrunken. With the time of exposure elongated , damage of cells became more severe.Conclusions: Four kinds of soft contact lens multi-purpose care solutions may have harmful effects on the culture of human corneal epithelial cells. Soaked lenses should be rinsed with saline before being placed in the eyes in order to reduce the potential toxicity of contact lens care solutions. Eye science 1998; 14 :45 - 47.
基金supported by grants from the National Natural Science Foundation of China(No. 81373269)CAMS Innovation Fund for Medical Sciences(No. 2016-12M-1-010)National Science and Technology Project of China(No.2017ZX09305008002)
文摘In this paper, quaternary 8-(1-acylethene-1-yl)-13-methylcoptisine chlorides targeting thioredoxin reductases(TrxRs) were designed to test the growth inhibitory activity against human cancer cell lines and the effect on viability of the normal intestinal epithelial cell-6(IEC-6) in vitro and to evaluate structure-activity relationship(SAR). The introduced α, β-unsaturated ketone groups at C-8 consisting of n-alkanoyls possessing five to ten carbons or aroyls or cyclohexylcarbonyl increased the tested activity against the target cancer cell lines. By and large, this type of improvement was increasingly graced by the elongation of the aliphatic chain of the n-alkanoyls in the range of less than ten carbon atoms. The relatively more polar 1-acylethene-1-yls displayed no effect on improving the activity. All the explored aroyls showed significant effect on improving the activity of the target compounds against the tested cancer cell lines with no SAR being observed. The findings of this study suggested that oil/water partition coefficient of the test compounds was one of the key factors impacting the target activity against the tested cancer cell lines. At the concentration of 10 μmol/L, except for the compounds with n-alkanoyls possessing seven or more carbons or with α-naphthoyl, none of the other compounds displayed obvious cytotoxicity on normal IEC-6 cell when co-incubated. The survival rate of IEC-6 cell ranged from 75% to 100% for the noncytotoxic compounds.