SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo

AIM:To explore the effect of silent information regulator factor 2-related enzyme 1(SIRT1)on modulating apoptosis of human lens epithelial cells(HLECs)and alleviating lens opacification of rats through suppressing endoplasmic reticulum(ER)stress.METHODS:HLECs(SRA01/04)were treated with varying concentrations of tunicamycin(TM)for 24h,and the expression of SIRT1 and C/EBP homologous protein(CHOP)was assessed using real-time quantitative polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8(CCK-8)assay,respectively.In the SRA01/04 cell apoptosis model,which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation,the expression levels of SIRT1,CHOP,glucose regulated protein 78(GRP78),and activating transcription factor 4(ATF4)were examined.The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid(4-PBA;an ER stress inhibitor)was investigated.In vivo,age-related cataract(ARC)rat models were induced by sodium selenite injection,and the protective role of SIRT1,activated by SRT1720 intraperitoneal injections,was evaluated through morphology observation,hematoxylin and eosin(H&E)staining,Western blotting,and RT-PCR.RESULTS:SIRT1 expression was downregulated in TMinduced SRA01/04 cells.Besides,in SRA01/04 cells,both cell apoptosis and CHOP expression increased with the rising doses of TM.ER stress was stimulated by TM,as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model.Inhibition of SIRT1 by siRNA knockdown increased ER stress activation,whereas SRT1720 treatment had opposite results.4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis.In vivo,SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models.CONCLUSION:SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress.These findings suggest a novel strategy for cataract treatment focused on targeting ER stress,highlighting the therapeutic potential of SIRT1 modulation in ARC development.

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense

AIM:To determine whether an antisense RNA corresponding to the human Alu transposable element(Aluas RNA)can protect human lens epithelial cells(HLECs)from methylglyoxal-induced apoptosis.METHODS:Cell counting kit-8(CCK-8)and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays were used to assess HLEC viability.HLEC viability/death was detected using a Calcein-AM/PI double staining kit;the annexin V-FITC method was used to detect HLEC apoptosis.The cytosolic reactive oxygen species(ROS)levels in HLECs were determined using a reactive species assay kit.The levels of malondialdehyde(MDA)and the antioxidant activities of total-superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)were assessed in HLECs using their respective kits.RT-q PCR and Western blotting were used to measure m RNA and protein expression levels of the genes.RESULTS:Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxalinduced decrease in Bcl-2 m RNA and the methylglyoxalinduced increase in Bax m RNA.In addition,Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression.Aluas RNA inhibited the production of ROS induced by methylglyoxal,restored T-SOD and GSHPx activity,and moderated the increase in MDA content after treatment with methylglyoxal.Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes,including glutathione peroxidase,heme oxygenase 1,γ-glutamylcysteine synthetase and quinone oxidoreductase 1.Aluas RNA ameliorated methylglyoxal-induced increases of the m RNA and protein expression of Keap1 that is the negative regulator of Nrf2.CONCLUSION:Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Role of reactive oxygen species in epithelial-mesenchymal transition and apoptosis of human lens epithelial cells

AIM:To investigate the role of reactive oxygen species(ROS)in epithelial–mesenchymal transition(EMT)and apoptosis of human lens epithelial cells(HLECs).METHODS:Flow cytometry was used to assess ROS production after transforming growth factorβ2(TGF-β2)induction.Apoptosis of HLECs after H_(2)O_(2) and TGF-β2 interference with or without ROS scavenger N-acetylcysteine(NAC)were assessed by flow cytometry.The corresponding protein expression levels of the EMT markerα-smooth muscle actin(α-SMA),the extracellular matrix(ECM),marker fibronectin(Fn),and apoptosis-associated proteins were detected by using Western blotting in the presence of an ROS scavenger(NAC).Wound-healing and Transwell assays were used to assess the migration capability of HLECs.RESULTS:TGF-β2 stimulates ROS production within 8h in HLECs.Additionally,TGF-β2 induced HLECs cell apoptosis,EMT/ECM synthesis protein markers expression,and pro-apoptotic proteins production;nonetheless,NAC treatment prevented these responses.Similarly,TGF-β2 promoted HLECs cell migration,whereas NAC inhibited cell migration.We further determined that although ROS initiated apoptosis,it only induced the accumulation of the EMT markerα-SMA protein,but not COL-1 or Fn.CONCLUSION:ROS contribute to TGF-β2-induced EMT/ECM synthesis and cell apoptosis of HLECs;however,ROS alone are not sufficient for EMT/ECM synthesis.

Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose

AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are regulated,and whether this pathway could be specifically targeted.METHODS:hRPE cells were cultured in normal glucose or high glucose for 1,3,or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species(ROS)levels using a dihydroethidium kit.miR-27b-3p,Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunocytofluorescence(ICF),respectively.Western blot analyses were performed to determine nuclear and total Nrf2 protein levels.Nrf2,NQO1,and HO-1 expression levels by RT-qPCR,ICF,or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection.Finally,the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine.RESULTS:Persistent exposure to high glucose gradually suppressed hRPE Nrf2,NQO1,and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels.High glucose also promoted ROS release and inhibited cellular proliferation.Nrf2,NQO1,and HO-1 mRNA levels decreased after miR-27b-3p overexpression and,conversely,both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor.After treating hRPE cells exposed to high glucose with pyridoxamine,ROS levels tended to decreased,proliferation rate increased,Nrf2,NQO1,and HO-1 mRNA and protein levels were upregulated,and miR-27b-3p mRNA levels were suppressed.CONCLUSION:Nrf2 is a downstream target of miR-27b-3p.Furthermore,the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

Establishment of an untransfected human corneal epithelial cell line and its biocompatibility with denuded amniotic membrane

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 p 1 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.

Human amniotic epithelial cell transplantation for the repair of injured brachial plexus nerve: evaluation of nerve viscoelastic properties

The transplantation of embryonic stem cells can effectively improve the creeping strength of nerves near an injury site in animals. Amniotic epithelial cells have similar biological properties as em-bryonic stem cells; therefore, we hypothesized that transplantation of amniotic epithelial cells can repair peripheral nerve injury and recover the creeping strength of the brachial plexus nerve. In the present study, a brachial plexus injury model was established in rabbits using the C6root avulsion method. A suspension of human amniotic epithelial cells was repeatedly injected over an area 4.0 mm lateral to the cephal and caudal ends of the C6 brachial plexus injury site （1 × 106 cells/mL, 3μL/injection, 25 injections） immediately after the injury. The results showed that the decrease in stress and increase in strain at 7,200 seconds in the injured rabbit C6 brachial plexus nerve were mitigated by the cell transplantation, restoring the viscoelastic stress relaxation and creep properties of the brachial plexus nerve. The forepaw functions were also signiifcantly improved at 26 weeks after injury. These data indicate that transplantation of human amniotic epithelial cells can effec-tively restore the mechanical properties of the brachial plexus nerve after injury in rabbits and that viscoelasticity may be an important index for the evaluation of brachial plexus injury in animals.

MicroRNA-34a promoting apoptosis of human lens epithelial cells through down-regulation of B-cell lymphoma-2 and silent information regulator

AIM： To investigate the role of micro RNA-34a（mi R-34a） in the induction of apoptosis of human lens epithelial（HLE-B3） cells. METHODS： The apoptosis of HLE-B3 cells was detected by Annexin V-PE apoptosis detection kit after the treatment with 200 μmol/L H2O2 for 24h and lentiviral mi R-34 a vector transfection. The expression of mi R-34 a in the cells was quantified by quantitative real time polymerase chain reaction（q RT-PCR） in response to H2O2 exposure and the vector transfection. The effects of overexpression of mi R-34 a on the expression of B-cell lymphoma-2（Bcl-2） and silent information regulator 1（SIRT1） was determined by q RT-PCR and Western blot. RESULTS： The expression of mi R-34 a was up-regulated by the treatment of H2O2 in HLE-B3 cells. The increased expression of mi R-34 a is accompanied with the cell apoptosis. Consistence with the H2O2 exposure,ectopic overexpression of mi R-34 a in HLE-B3 cells promoted cells apoptosis. Importantly the anti-apoptosis factors Bcl-2 and SIRT1 were reduced significantly by up-regulation of mi R-34 a in HLE-B3 cells.CONCLUSION： Mi R-34 a promotes the apoptosis of HLE-B3 cells by down-regulating Bcl-2 and SIRT1,suggesting that mi R-34 a may involve in the pathogenesis of cataract formation and targeting mi R-34 a may be a potentially therapeutic approach for treatment of cataract.

Monoamine alterations and rotational asymmetry in a rat model of Parkinson's disease following lateral ventricle transplantation of human amniotic epithelial cells

BACKGROUND： Human amniotic epithelial cells （HAECs） can differentiate into neurons, astrocytes and oligodendrocytes. They biologically secrete many active neurotrophins and have the capacity to metabolize dopamine enzymes. These features underlie a theoretical basis for the treatment of Parkinson＇s disease （PD）. OBJECTIVE： To investigate the survival and differentiation of transplanted HAECs in the lateral ventricle of PD model rats, and to explore its effect on circling behavior, as well as levels of dopamine （DA）, the metabolite homovanillic acid, dihydroxyphenyl acetic acid, 5-hydroxyindoleacetic acid, and 5-hydroxytryptamine in the striatum. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, and Shanghai Celstar Institute of Biotechnology from May 2007 to December 2008. MATERIALS： HAECs were derived from the placental chorion following caesarean delivery at the Shanghai International Matemal and Child Health Hospital. 6-hydroxydopamine （6-OHDA）, and mouse anti-human Vimentin monoclonal antibody were purchased from Sigma, USA; mouse anti-human nestin and tyrosine hydroxylase （TH） monoclonal antibodies were purchased from Chemicon, USA. METHODS： A total of 114 healthy, adult, Sprague Dawley rats were randomly assigned to two groups： PD model [n = 90, stereotactic microinjection of 2 μL 6-OHDA （3.5 μg/uL） into the striatum] and control （n = 24, no treatment）. The 51 successful PD model rats were randomly divided into 3 subgroups （n = 17）： HAEC, PBS, and model. The HAEC and PBS groups were respectively injected with 10 μL PBS solution containing 1 × 10^5/mL HAECs or 10 pL PBS into the lateral ventricle. The model group was not treated. MAIN OUTCOME MEASURES： TH protein expression in the striatum was evaluated by immunohistochemistry 5 weeks after HAEC transplantation. At 10 weeks, HAEC survival in the lateral ventricle was investigated by immunofluorescent staining; differentiation of HAECs in the lateral and third ventricles was examined by TH immunohistochemistry; concentrations of DA, homovanillic acid, dihydroxyphenyl acetic acid, 5-hydroxyindoleacetic acid, and 5-hydroxytryptamine in the striatum, as well as DA concentration in the cerebrospinal fluid, were measured with high-performance liquid chromatography-electrochemical detection. Circling behavior of PD model rats was consecutively observed for 10 weeks following intraperitoneal injection of amphetamine 1 week after successful model establishment. RESULTS： tn the HAEC group, the number of TH-positive cells significantly increased in the striatum, and circling behavior significantly decreased, compared with the PBS and model groups （P 〈 0.01）. In addition, monoamine concentrations in the striatum, as well as DA concentrations in the cerebrospinal fluid, significantly increased, compared with the PBS group （P 〈 0.05-0.01）. Moreover, a large number of nestin-, vimentin-, and TH-positive cells were observed in the lateral and third ventricles following HAEC injection.CONCLUSION： HAECs survived for 10 weeks with no overgrowth following transplantation into the lateral ventricle of PD model rats. Moreover, the cells differentiated into dopaminergic neurons, which increased DA secretion. HAEC transplantation improved cycling behavior in PD model rats.

Expression of dectin-1 during fungus infection in human corneal epithelial cells

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.

Effects of Rapamycin on Expression of Bcl-2 and Bax in Human Lens Epithelial Cells and Cell Cycle in Rats

The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells（LECs） and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract.The cultured LECs of second and third passages were collected and treated with rapamycin.The LECs were transferred into 96-well culture plates and divided into 6 groups,and each group was set to have 8 duplicate wells.In the negative control group,the LECs were given culture medium only,and in the blank control group,only culture medium was given.In the four rapamycin-treated groups,different concentrations（20,40,60 and 80 ng/mL） of rapamycin were given.After treatment for 24,48 and 72 h,the absorbance（A） values in each well were determined by MTT assay.The cell cycles of all groups were detected by using flow cytometry.Real-time fluorescent quantitative polymerase chain reaction（RFQ-PCR） and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively.MTT assay showed that rapamycin could inhibit proliferation of LECs in a time-and dose-dependent manner.Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase,resulting in the increase of cells in G1 phase and the decrease of the cells in S phase.RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA,but up-regulate the expression of Bax mRNA,suggesting it could induce apoptosis of LECs.Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein,but promote the expression of Bax protein.It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle,but also by promoting the induction of apoptosis.

In vitro inhibition of proliferation,migration and epithelial-mesenchymal transition of human lens epithelial cells by fasudil

AIM： To study the potential role of fasudil as a treatment for posterior capsular opacification（PCO） of the human crystalline lens.METHODS： Human lens epithelial cells（HLECs; line SRA01/04） was exposed to transforming growth factor-β2（TGF-β2） to induce the process of epithelial-mesenchymal transition（EMT）. Fasudil was applied to the cell samples. Its effect on overall HLECs proliferation and migration was studied, as was its influence on EMT induction by TGF-β2 using cell migration assay, MTT colorimetric assay and Western blot assay.RESULTS： Fasudil inhibited the proliferation of SRA01/04. Its effect was time-and concentration-dependent. The migration of SRA01/04 cells was significantly reduced 24-72 h after fasudil treatment, and the half maximal inhibitory concentration（IC50） was 22.37 μmol/mL at 72 h. Reversal of the elongated, fibroblast-like shape changes induced by TGF-β2 in SRA01/04 cells was observed. Fasudil up-regulated the expression of Connexin43 protein and down-regulated the expression of α-SMA protein compared with the cells treated with TGF-β2. Furthermore, when exposed to fasudil, the phosphorylation of Rhoassociated protein kinase（Rock） and myosin light chain（MLC） could not be activated in the cell preparations.CONCLUSION： Fasudil suppresses the proliferation and migration of SRA01/04 cells, and inhibits the process of EMT induced by TGF-β2. These results suggest that fasudil may serve as a therapeutic agent for PCO.

Effects of Sodium Salicylate on the Expression of HSP27 Protein during Oxidative Stress in Tissue-cultured Human Lens Epithelial Cells

The effects of sodium salicylate on the expression of heat shock protein 27 （HSP27） during oxidative stress in tissue-cultured human lens epithelial cells were investigated. Cultured human lens epithelial cells （HLB-3） were divided into 3 groups： control group （group A）, oxidation injury group （group B） and sodium salicylate group （group C）. Apoptosis of human lens epithelial cells cultured in vitro was induced in the presence of 150 μmol/L H2O2. Cells viability and the expression of HSP27 were analyzed. Viability of the cells was measured by methyl thiazole tetrazolium （MTT） chromatometry. The expression of HSP27 in HLB-3 cells was detected by using immunohistochemistry and image analysis system, Sodium salicylate could induce the expression of HSP27, and the cells viability in group C was significantly higher than in group B （0.2667±0.01414 vs 0.2150±0.01080, P=0.012〈0.05）. The average gray value of HSP27 in group B was less than that in group C （P=0.000〈0.05）. The increased expression of HSP27 by sodium salicylate might play an important role in the protection of hydrogen peroxide-induced injury of human lens epithelial cells, suggesting that sodium salicylate could suppress, at least in part, the apoptosis of human lens epithelial cells.

Human amniotic epithelial cells express specific markers of nerve cells and migrate along the nerve fibers in the corpus callosum

Human amniotic epithelial cells were isolated from a piece of fresh amnion. Using immunocytochemical methods, we investigated the expression of neuronal phenotypes （microtubule-associated protein-2, glial fibrillary acidic protein and nestin） in human amniotic epithelial cells. The conditioned medium of human amniotic epithelial cells promoted the growth and proliferation of rat glial cells cultured in vitro, and this effect was dose-dependent. Human amniotic epithelial cells were further transplanted into the corpus striatum of healthy adult rats and the grafted cells could integrate with the host and migrate 1 2 mm along the nerve fibers in corpus callosum. Our experimental findings indicate that human amniotic epithelial cells may be a new kind of seed cells for use in neurograft.

Quercetin exerts anti-inflammatory effects via inhibiting tumor necrosis factor-α-induced matrix metalloproteinase-9 expression in normal human gastric epithelial cells

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.

Transplantation of human amniotic epithelial cells repairs brachial plexus injury:pathological and biomechanical analyses

A brachial plexus injury model was established in rabbits by stretching the C6 nerve root. Imme- diately after the stretching, a suspension of human amniotic epithelial cells was injected into the injured brachial plexus. The results of tensile mechanical testing of the brachial plexus showed that the tensile elastic limit strain, elastic limit stress, maximum stress, and maximum strain of the injured brachial plexuses were significantly increased at 24 weeks after the injection. The treatment clearly improved the pathological morphology of the injured brachial plexus nerve, as seen by hematoxylin eosin staining, and the functions of the rabbit forepaw were restored. These data indicate that the injection of human amniotic epithelial cells contributed to the repair of brachial plexus injury, and that this technique may transform into current clinical treatment strategies.

In vitro reconstruction and characterization of tissue-engineered human corneal epithelium with seeder cells from an untransfected human corneal epithelial cell line

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 beta 1) 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.

The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

This study aimed to compare epithelial cells derived from human embryonic stem cells(hESCs) to human ameloblast-lineage cells (ALCs),as a way to determine their potential use as a cell source for ameloblast regeneration.Induced by various concentrations of bone morphogenetic protein 4(BMP4),retinoic acid(RA) and lithium chloride(LiCI) for 7 days,hESCs adopted cobble-stone epithelial phenotype(hESC-derived epithelial cells(ES-ECs)) and expressed cytokeratin 14.Compared with ALCs and oral epithelial cells(OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs.ES-ECs were compared with human fetal skin epithelium,human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well.ALCs had relatively high expression levels of cytokeratin 76,which was also found to be upregulated in ES-ECs.Based on the present study,with the similarity of gene expression with ALCs,ES-ECs are a promising potential cell source for regeneration,which are not available in erupted human teeth for regeneration of enamel.

Alterative Expression and Sequence of Human Elongation Factor-1δ during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.

Possible Role of DNA Polymerase beta in Protecting Human Bronchial Epithelial Cells Against Cytotoxicity of Hydroquinone

Objective To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. Methods DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone （ranged from 10 μmol/L to 120 μmol/L） for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis （SCGE）] were performed respectively to detect the toxicity of hydroquinone. Results assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. Conclusions Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells

AIM：To investigate the effects of lentivirus（LV）mediated integrin-linked kinase（ILK）RNA interference（RNAi）on biological behaviors of human lens epithelial cells（LECs）.·METHODS：Human cataract LECs and immortalized human LEC line,human lens epithelial（HLE）B-3 cells were transfected by lentiviral vector expressing ILKspecific short hairpin RNA（sh RNA）and then stimulated by transforming growth factor-β（TGF-β）,the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction（RT-PCR）and Western blot methods;biological behaviors including cell cycle and apoptosis,cell morphology,α-smooth muscle actin（SMA）stress fiber formation and cell migration were examined.·RESULTS：Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-sh RNA vector;flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells.Lessα-SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs.·CONCLUSION：The present study demonstrated that ILK was an important regulator for LECs proliferation and migration.LV mediated ILK RNAi is an effective way todecrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis,as well as,to prevent cell migration by inhibiting TGF-βinducedα-SMA stress fiber formation.Thus,LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.