In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of A...In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.展开更多
AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE ce...AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).展开更多
AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypox...AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2.The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank.The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software.The small interfering RNA(siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM,and transfected into the hypoxic hepatoma cells.Real time reverse transcription-polymerase chain reaction(RTPCR) and Western blotting assay were used to detect the expression levels of mRNA and protein.HIF-1α and vascular endothelial growth factor(VEGF) mRNA was determined using real time RT-PCR;the protein expression levels of AKT,p-AKT,p21 and cyclinD1 were determined using Western blotting.The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium(MTT) assay and the bromodeoxyuridine(BrdU) incorporation cell proliferation assay.RESULTS:Under induced hypoxia,the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2;the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L.Under hypoxia,the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia.HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells.The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF,along with the protein expression levels of p-AKT and cyclinD1;the protein expression of p21 was significantly increased,and there was no significant difference in the expression of AKT.The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group;in the siRNA transfection group,the amount of hepatoma cells in G1 phase was more than that in the control group.The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group.The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells.CONCLUSION:Hypoxia increases the expression of HIF-1α,and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.展开更多
Background: In patients with difuse large B?cell lymphoma(DLBCL), central nervous system(CNS) relapse is uncom?mon but is nearly always fatal. This study aimed to determine the risk factors for CNS relapse in DLBCL pa...Background: In patients with difuse large B?cell lymphoma(DLBCL), central nervous system(CNS) relapse is uncom?mon but is nearly always fatal. This study aimed to determine the risk factors for CNS relapse in DLBCL patients and to evaluate the eicacy of rituximab and intrathecal chemotherapy prophylaxis for CNS relapse reduction.Methods: A total of 511 patients with newly diagnosed DLBCL treated at the Sun Yat?sen University Cancer Center between January 2003 and December 2012 were included in the study. Among these patients, 376 received R?CHOP regimen(rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) as primary treatment, and 135 received CHOP regimen(cyclophosphamide, doxorubicin, vincristine, and prednisone) as primary treatment. Intrathe?cal chemotherapy prophylaxis(methotrexate plus cytarabine) was administered to those who were deemed at high risk for CNS relapse. In the entire cohort and in the R?CHOP set in particular, the Kaplan–Meier method coupled with the log?rank test was used for univariate analysis, and the Cox proportional hazards model was used for multivariate analysis. Diferences were evaluated using a two?tailed test, and P < 0.05 was considered signiicant.Results: At a median follow?up of 46 months, 25(4.9%) patients experienced CNS relapse. There was a trend of reduced occurrence of CNS relapse in patients treated with rituximab; the 3?year cumulative CNS relapse rates were 7.1% in CHOP group and 2.7% in R?CHOP group(P = 0.045). Intrathecal chemotherapy prophylaxis did not confer much beneit in terms of preventing CNS relapse. Bone involvement [hazard ratio(HR) = 4.21, 95% conidence interval(CI) 1.38–12.77], renal involvement(HR = 3.85, 95% CI 1.05–14.19), alkaline phosphatase(ALP) >110 U/L(HR = 3.59, 95% CI 1.25–10.34), serum albumin(ALB) <35 g/L(HR = 3.63, 95% CI 1.25–10.51), treatment with rituxi?mab(HR = 0.34, 95% CI 0.12–0.96), and a time to complete remission ≤ 108 days(HR = 0.22, 95% CI 0.06–0.78) were independent predictive factors for CNS relapse in the entire cohort. Bone involvement(HR = 4.44, 95% CI 1.08–18.35), bone marrow involvement(HR = 11.70, 95% CI 2.24–60.99), and renal involvement(HR = 10.83, 95% CI 2.27–51.65) were independent risk factors for CNS relapse in the R?CHOP set.Conclusions: In the present study, rituximab decreased the CNS relapse rate of DLBCL, whereas intrathecal chemo?therapy prophylaxis alone was not suicient for preventing CNS relapse. Serum levels of ALB and ALP, and the time to complete remission were new independent predictive factors for CNS relapse in the patients with DLBCL. In the patients received R?CHOP regimen, a trend of increased CNS relapse was found to be associated with extranodal lesions.展开更多
AIMTo expose rat retinal Müller cells to 530 nm monochromatic light and investigate the influence of varying light illumination times on basic fibroblast growth factor (bFGF) and transforming growth factor...AIMTo expose rat retinal Müller cells to 530 nm monochromatic light and investigate the influence of varying light illumination times on basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) expression.METHODSThree groups of rat retinal Müller cells cultured in vitro under a 530 nm monochromatic light were divided into 6, 12 and 24h experimental groups, while cells incubated under dark conditions served as the control group. The bFGF and TGF-β1 mRNA expression, protein levels and fluorescence intensity of the Müller cells were analyzed.RESULTSThe bFGF mRNA expression and protein levels were significantly upregulated in Müller cells in all three experimental groups compared with the control group (P<0.05), while that of TGF-β1 was downregulated (P<0.05). Also, bFGF expression was positively correlated, but TGF-β1 expression was negatively correlated with illumination time. The largest changes for both cytokines were seen in the 24h group. The changes in bFGF and TGF-β1 fluorescence intensity were highest in the 24h group, and significant differences were observed among the experimental groups (P<0.05).CONCLUSIONThe expressions of bFGF and TGF-β1 changed in a time-dependent manner in Müller cells exposed to 530 nm monochromatic light with 250 lx illumination intensity. Müller cells might play a role in the development of myopia by increasing bFGF expression or decreasing TGF-β1 expression. Changes in cytokine expression in retinal Müller cells may affect monochromatic light-induced myopia.展开更多
Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, a...Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, and the effect of NF-κB activation on the protection of Parkinson’s disease by Isoflavone (I). Methods: PC12 cells were used to establish the cell model of Parkinson’s disease, and are divided into five groups: control group;MPP+ group;I (Isoflavone) + MPP+ group;I group;SN-50 + MPP+ group. The content of NF-κB in PC12 cells was determined by immunocytochemistry;The viability of PC12 cells after treated with cell-permeable NF-κB inhibitor SN-50 and cell viability were measured by MTT assay;the expression levels of NF-κB p65 in cytoplasm and nuclear fractions were evaluated by western blot analysis;the mRNA expression of NF-κB p65 was analyzed by in situ hybridization (ISH). Results: Compared with the control group, the protein of NF-κB p65 both in cytoplasm and in nuclei was significantly higher than in I + MPP+ and MPP+ groups;similarly, the mRNA expression level of NF-κB p65 gene was also significantly higher;moreover, the protein expression of NF-κB p65 was much lower in I group (P + group, the protein of NF-κB p65 was significantly lower in I + MPP+ group, the mRNA expression level of NF-κB p65 gene was also significantly lower, and the protein expression level of NF-κB p65 was much lower in I + MPP+ group (P + group (P > 0.05). Conclusion: NF-κB activation is essential to MPP+-induced apoptosis in PC12 cells;but Isoflavone can inhibit the cell damage to some extent to execute its protective function, which may be involved in nigral neurodegeneration in patients with Parkinson’s disease.展开更多
Objective: To investigate the role of peroxisome proliferator-activated receptors δ (PPARδ) in inflammatory reaction and its possible mechanism in adipocyte. Methods:Lentivirus-mediated RNA interference (RNAi)...Objective: To investigate the role of peroxisome proliferator-activated receptors δ (PPARδ) in inflammatory reaction and its possible mechanism in adipocyte. Methods:Lentivirus-mediated RNA interference (RNAi) was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α(TNFα, 20 ng/ml) for 4 h. The expression of PPARδ, nuclear factor κB (NFκB) and C reactive protein (CRP) were determined by Western blot analysis. Results:The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion: PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.展开更多
BACKGROUND Type 2 diabetes(T2 D) is characterized by insufficient insulin secretion caused by defective pancreatic β-cell function or insulin resistance,resulting in an increase in blood glucose.However,the mechanism...BACKGROUND Type 2 diabetes(T2 D) is characterized by insufficient insulin secretion caused by defective pancreatic β-cell function or insulin resistance,resulting in an increase in blood glucose.However,the mechanism involved in this lack of insulin secretion is unclear.The level of vascular endothelial growth factor B(VEGF-B) is significantly increased in T2 D patients.The inactivation of VEGF-B could restore insulin sensitivity in db/db mice by reducing fatty acid accumulation.It is speculated that VEGF-B is related to pancreatic β-cell dysfunction and is an important factor affecting β-cell secretion of insulin.As an in vitro model of normal pancreatic β-cells,the MIN6 cell line can be used to analyze the mechanism of insulin secretion and related biological effects.AIM To study the role of VEGF-B in the insulin secretion signaling pathway in MIN6 cells and explore the effect of VEGF-B on blood glucose regulation.METHODS The MIN6 mouse pancreatic islet β-cell line was used as the model system.By administering exogenous VEGF-B protein or knocking down VEGF-B expression in MIN6 cells,we examined the effects of VEGF-B on insulin secretion,Ca2+ and cyclic adenosine monophosphate(cAMP) levels,and the insulin secretion signaling pathway.RESULTS Exogenous VEGF-B inhibited the secretion of insulin and simultaneously reduced the levels of Ca2+ and cAMP in MIN6 cells.Exogenous VEGF-B also reduced the expression of phospholipase C gamma 1(PLCγ1),phosphatidylinositol 3-kinase(PI3 K),serine/threonine kinase(AKT),and other proteins in the insulin secretion pathway.Upon knockdown of VEGF-B,MIN6 cells exhibited increased insulin secretion and Ca2+ and cAMP levels and upregulated expression of PLCγ1,PI3 K,AKT,and other proteins.CONCLUSION VEGF-B can regulate insulin secretion by modulating the levels of Ca2+ and cAMP.VEGF-B involvement in insulin secretion is related to the expression of PLCγ1,PI3 K,AKT,and other signaling proteins.These results provide theoretical support and an experimental basis for the study of VEGF-B in the pathogenesis of T2 D.展开更多
Objective: To assess the clinical features, survival and prognostic factors of primary testicular diffuse large B-cell lymphoma (DLBCL). Methods: A retrospective study of 37 patients with primary testicular DLBCL ...Objective: To assess the clinical features, survival and prognostic factors of primary testicular diffuse large B-cell lymphoma (DLBCL). Methods: A retrospective study of 37 patients with primary testicular DLBCL was carried out from November 2003 to May 2012. Their clinical features, survival and prognostic factors were analyzed. Results: During a median follow-up period of 39.8 months (5.4-93.0 months), the median progression-free survival (PFS) was 26.2 months (95% CI:0-65 months) and the 3-year overall survival (OS) rate was 78.4%. Within the whole cohort, the factors significantly associated with a superior PFS were limited stage (stage Ⅰ/Ⅱ), lactate dehydrogenase (LDH) ≤245 U/L, international prognostic index (IPI) ≤1, primary tumor diameter 〈7.5 cm, and patients who had complete response (CR) and received doxoruhicin-contained chemotherapy (P〈0.05). There was a trend toward superior outcome for patients who received combined therapy (surgery/ chemotherapy/radiotherapy) (P=0.055). Patients who had CR, primary tumor diameter 〈7.5 cm and IPI score ≤1 were significantly associated with longer PFS at multivariate analysis. Conclusions: Primary testicular DLBCL had poorer survival. CR, primary tumor diameter and IPI were independent prognostic factors. The combined therapy of orchectomy, doxorubicin-contained chemotherapy and contralateral testicular radiotherapy (RT) seemed to improve survival.展开更多
A human B cell line (3D5) that responds specifically to B cell growth factor (BCGF) hasbeen developed by a sequence of Staphylococcus aureus Cowen I activation,EB virus im-mortalization,and cloning.Proliferative r...A human B cell line (3D5) that responds specifically to B cell growth factor (BCGF) hasbeen developed by a sequence of Staphylococcus aureus Cowen I activation,EB virus im-mortalization,and cloning.Proliferative response to PHA-stimulated T cell supernatant(PHA-T-Sup) and nonresponsiveness to rIL-2 stimulation were factors used to screen positivecells.Phenotype analysis with a flow cytometer indicated that:1) 3D5 is a B cell line:100% of the cells were positive for B1 marker and 59% were positive for sIg,while T3and Mo 1 were negative:2) 3D5 is an activated B cell line:both Tac and 4F2 markersof activated (but not of resting) B cells were 100% positive:3) 3D5 expresses high molecularweight BCGF (HMW-BCGF) receptor-associated epitope BA5.3D5 cells proliferated inresponse to cpBCGF stimulation in a dose-dependent manner.HMW-BCGF also induced3D5 cells to proliferate.Interestingly.no proliferation could be detected in the presenceof rIL-2,rIL-4,or rIFN-r.The data show that 3D5 cells are specifically BCGF-responsiveB cells.Using 3D5 cells as target,BCGF activity was detected in crude BCGF preparationsedimented by 85% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> and chromatographed in a DEAE-Sephadex A-25 column fromPHA-T-Sup.T24 cell supernatant with B cell differentiation factor (BCDF) activity couldnot induce 3D5 cells to differentiate into immunoglobulin-secreting cells.展开更多
AIM:To study the inhibition of nuclear factor kappa-B p65(NF-κB p65)antisense oligodeoxynucleotide(ASODN)on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2(T...AIM:To study the inhibition of nuclear factor kappa-B p65(NF-κB p65)antisense oligodeoxynucleotide(ASODN)on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2(TGF-β2).·M ETHODS:NF-κBp65ASODNand NF-κBp65missense oligodeoxynucleotide(MSODN)were designed and synthesized.Human lens epithelial cell line(HLE B-3)cells were prepared for study and divided into 7 groups.Control group was HLE B-3 cells cultured in dulbecco’s modified eagle medium(DMEM).T1,T2,and T3 group were HLE B-3 cells cultured in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A+T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M+T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h.The negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent(Hi Per Fect)for 24h.Cell morphology was observed at different time points using an inverted microscope.The expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction(RT-PCR),and the expression ofα-smooth muscle actin(α-SMA)protein was assayed with ELISA.·RESULTS:With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant(〈0.05).NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.NF-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.The difference between control group and A+T group was not statistically significant(〉0.05),but the difference among A+T group and other groups was statistically significant(〈0.05).·CONCLUSION:NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiation of HLE B-3.NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.展开更多
The relation between the expression and activity of MMP-9 in C-reactive protein (CRP)-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B (NF-κB) was studied to investigate the poss...The relation between the expression and activity of MMP-9 in C-reactive protein (CRP)-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B (NF-κB) was studied to investigate the possible role of CRP in plaque destabilization. Human THP-1 cells were incubated in the presence of CRP at 0 (control group), 25, 50 and 100 μg/mL (CRP groups) for 24 h. In PDTC (a specific NF-κB inhibitor) group, the cells were pre-treated with PDTC at 10 μmol/L and then with 100 μg/mL CRP. The conditioned media (CM) and human THP-1 cells in different groups were harvested. MMP-9 expression in CM and human THP-1 cells was measured by ELISA and Western blotting. MMP-9 activity was assessed by fluorogenic substrates. The expression of NF-κB inhibitor α (IκB-α) and NF-κB p65 was detected by Western blotting and ELISA respectively. The results showed that CRP increased the expression and activity of MMP-9 in a dose-dependent manner in the human THP-1 cells. Western blotting revealed that IiB-α expression was decreased in the cells with the concentrations of CRP and ELISA demonstrated that NF-κB p65 expression in the CRP-induced cells was increased. After pre-treatment of the cells with PDTC at 10 μmol/L, the decrease in IκB-α expression and the increase in NF-κB p65 expression in the CRP-induced cells were inhibited, and the expression and activity of MMP-9 were lowered too. It is concluded that increased expression and activity of MMP-9 in CRP-induced human THP-1 cells may be associated with activation of NF-κB. Down-regulation of the expression and activity of MMP-9 may be a new treatment alternative for plaque stabilization by inhibiting the NF-κB activation.展开更多
By study on the effect of anisodamine on lipopolysaccharide- induced expression of tissue factor(TF) in vascular endothelial cells(EC) ,the mechanism of anisodam ine antithrom bosis,as well as in the treatm ent of b...By study on the effect of anisodamine on lipopolysaccharide- induced expression of tissue factor(TF) in vascular endothelial cells(EC) ,the mechanism of anisodam ine antithrom bosis,as well as in the treatm ent of bacteraemic shock was investigated.Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion m ethod.TF activity was measured in the lysates of HUVEC by using a single step clotting assay.Specific m RNA expression was detected by Northern blotting.In order to evaluate a possible contribution of the nuclear factor (NF) -κB pathway on the effects observed,electrophoretic mobility shift assays(EMSA) were performed using nuclear extracts from HU VECs and NF- κB- binding oligonucleotides.The results showed that treatment of HUVEC with L PS resulted in a significant increase in TF activity.Anisodamine dose- dependently inhibited L PS- induced upregulation of TF.These effects was also confirm ed on the level of specific TF m RNA expression by Northern blotting.Furtherm ore,EMSA showed that anisodamine com pletely abolished L PS- induced NF-κB DNA binding activity in nuclear ex- tracts from HUVECs treated with L PS together with anisodamine.The results suggest thataniso- damine counteracts endothelial cell activation by inhibiting L PS- induced TF expression in these cells.Its interference with the NF- κB pathway might- at least in part- contribute to this effect. The ability of anisodamine to counteract L PS effect on endothelial cells m ight be one underlying m echanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.展开更多
Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering ...Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.展开更多
Objective: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymp...Objective: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1). Methods: A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni2+-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion: The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.展开更多
基金supported by STI2030-Major Projects,No.2021ZD 0201801(to JG)Shanxi Province Basic Research Program,No.20210302123429(to QS).
文摘In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.
基金Supported by the Natural Science Foundation of Shaanxi Province,China(No.2022JM-521).
文摘AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).
基金Supported by Natural Science Foundation of Guangdong Province People’s Republic of China,No. 10151008901000182
文摘AIM:To study the effects of hypoxia-inducible factor1α(HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.METHODS:The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2.The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank.The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software.The small interfering RNA(siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM,and transfected into the hypoxic hepatoma cells.Real time reverse transcription-polymerase chain reaction(RTPCR) and Western blotting assay were used to detect the expression levels of mRNA and protein.HIF-1α and vascular endothelial growth factor(VEGF) mRNA was determined using real time RT-PCR;the protein expression levels of AKT,p-AKT,p21 and cyclinD1 were determined using Western blotting.The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium(MTT) assay and the bromodeoxyuridine(BrdU) incorporation cell proliferation assay.RESULTS:Under induced hypoxia,the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2;the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L.Under hypoxia,the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia.HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells.The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF,along with the protein expression levels of p-AKT and cyclinD1;the protein expression of p21 was significantly increased,and there was no significant difference in the expression of AKT.The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group;in the siRNA transfection group,the amount of hepatoma cells in G1 phase was more than that in the control group.The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group.The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells.CONCLUSION:Hypoxia increases the expression of HIF-1α,and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.
基金supported by the National Natural Science Foundation of China(81372883,81001052)Natural Science Foundation of Guangdong Province,China(2015A030313020 and 8151008901000043)+3 种基金Science and Technology Planning Project of Guangdong Province,China(2011B031800222)Young Talents Key Project of Sun Yat?sen University(2015ykzd13,to Qing-qing Cai)Young Talents Project of Sun Yat-sen University(11ykpy56,to Qing-qing Cai)the Sister Institution Network Fund of MD Anderson Cancer Center(to Qing-qing Cai and Hui-Rao)
文摘Background: In patients with difuse large B?cell lymphoma(DLBCL), central nervous system(CNS) relapse is uncom?mon but is nearly always fatal. This study aimed to determine the risk factors for CNS relapse in DLBCL patients and to evaluate the eicacy of rituximab and intrathecal chemotherapy prophylaxis for CNS relapse reduction.Methods: A total of 511 patients with newly diagnosed DLBCL treated at the Sun Yat?sen University Cancer Center between January 2003 and December 2012 were included in the study. Among these patients, 376 received R?CHOP regimen(rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) as primary treatment, and 135 received CHOP regimen(cyclophosphamide, doxorubicin, vincristine, and prednisone) as primary treatment. Intrathe?cal chemotherapy prophylaxis(methotrexate plus cytarabine) was administered to those who were deemed at high risk for CNS relapse. In the entire cohort and in the R?CHOP set in particular, the Kaplan–Meier method coupled with the log?rank test was used for univariate analysis, and the Cox proportional hazards model was used for multivariate analysis. Diferences were evaluated using a two?tailed test, and P < 0.05 was considered signiicant.Results: At a median follow?up of 46 months, 25(4.9%) patients experienced CNS relapse. There was a trend of reduced occurrence of CNS relapse in patients treated with rituximab; the 3?year cumulative CNS relapse rates were 7.1% in CHOP group and 2.7% in R?CHOP group(P = 0.045). Intrathecal chemotherapy prophylaxis did not confer much beneit in terms of preventing CNS relapse. Bone involvement [hazard ratio(HR) = 4.21, 95% conidence interval(CI) 1.38–12.77], renal involvement(HR = 3.85, 95% CI 1.05–14.19), alkaline phosphatase(ALP) >110 U/L(HR = 3.59, 95% CI 1.25–10.34), serum albumin(ALB) <35 g/L(HR = 3.63, 95% CI 1.25–10.51), treatment with rituxi?mab(HR = 0.34, 95% CI 0.12–0.96), and a time to complete remission ≤ 108 days(HR = 0.22, 95% CI 0.06–0.78) were independent predictive factors for CNS relapse in the entire cohort. Bone involvement(HR = 4.44, 95% CI 1.08–18.35), bone marrow involvement(HR = 11.70, 95% CI 2.24–60.99), and renal involvement(HR = 10.83, 95% CI 2.27–51.65) were independent risk factors for CNS relapse in the R?CHOP set.Conclusions: In the present study, rituximab decreased the CNS relapse rate of DLBCL, whereas intrathecal chemo?therapy prophylaxis alone was not suicient for preventing CNS relapse. Serum levels of ALB and ALP, and the time to complete remission were new independent predictive factors for CNS relapse in the patients with DLBCL. In the patients received R?CHOP regimen, a trend of increased CNS relapse was found to be associated with extranodal lesions.
文摘AIMTo expose rat retinal Müller cells to 530 nm monochromatic light and investigate the influence of varying light illumination times on basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) expression.METHODSThree groups of rat retinal Müller cells cultured in vitro under a 530 nm monochromatic light were divided into 6, 12 and 24h experimental groups, while cells incubated under dark conditions served as the control group. The bFGF and TGF-β1 mRNA expression, protein levels and fluorescence intensity of the Müller cells were analyzed.RESULTSThe bFGF mRNA expression and protein levels were significantly upregulated in Müller cells in all three experimental groups compared with the control group (P<0.05), while that of TGF-β1 was downregulated (P<0.05). Also, bFGF expression was positively correlated, but TGF-β1 expression was negatively correlated with illumination time. The largest changes for both cytokines were seen in the 24h group. The changes in bFGF and TGF-β1 fluorescence intensity were highest in the 24h group, and significant differences were observed among the experimental groups (P<0.05).CONCLUSIONThe expressions of bFGF and TGF-β1 changed in a time-dependent manner in Müller cells exposed to 530 nm monochromatic light with 250 lx illumination intensity. Müller cells might play a role in the development of myopia by increasing bFGF expression or decreasing TGF-β1 expression. Changes in cytokine expression in retinal Müller cells may affect monochromatic light-induced myopia.
文摘Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, and the effect of NF-κB activation on the protection of Parkinson’s disease by Isoflavone (I). Methods: PC12 cells were used to establish the cell model of Parkinson’s disease, and are divided into five groups: control group;MPP+ group;I (Isoflavone) + MPP+ group;I group;SN-50 + MPP+ group. The content of NF-κB in PC12 cells was determined by immunocytochemistry;The viability of PC12 cells after treated with cell-permeable NF-κB inhibitor SN-50 and cell viability were measured by MTT assay;the expression levels of NF-κB p65 in cytoplasm and nuclear fractions were evaluated by western blot analysis;the mRNA expression of NF-κB p65 was analyzed by in situ hybridization (ISH). Results: Compared with the control group, the protein of NF-κB p65 both in cytoplasm and in nuclei was significantly higher than in I + MPP+ and MPP+ groups;similarly, the mRNA expression level of NF-κB p65 gene was also significantly higher;moreover, the protein expression of NF-κB p65 was much lower in I group (P + group, the protein of NF-κB p65 was significantly lower in I + MPP+ group, the mRNA expression level of NF-κB p65 gene was also significantly lower, and the protein expression level of NF-κB p65 was much lower in I + MPP+ group (P + group (P > 0.05). Conclusion: NF-κB activation is essential to MPP+-induced apoptosis in PC12 cells;but Isoflavone can inhibit the cell damage to some extent to execute its protective function, which may be involved in nigral neurodegeneration in patients with Parkinson’s disease.
文摘Objective: To investigate the role of peroxisome proliferator-activated receptors δ (PPARδ) in inflammatory reaction and its possible mechanism in adipocyte. Methods:Lentivirus-mediated RNA interference (RNAi) was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α(TNFα, 20 ng/ml) for 4 h. The expression of PPARδ, nuclear factor κB (NFκB) and C reactive protein (CRP) were determined by Western blot analysis. Results:The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion: PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.
基金Supported by National Natural Science Foundation of China,No.31771284National Natural Science Foundation of China Youth Project,No.31702024+1 种基金Major Basic Research Project of Shandong Provincial Natural Science Foundation,No.ZR2019ZD27Shandong Province Higher Educational Science and Technology Plan Project,No.J17KA258。
文摘BACKGROUND Type 2 diabetes(T2 D) is characterized by insufficient insulin secretion caused by defective pancreatic β-cell function or insulin resistance,resulting in an increase in blood glucose.However,the mechanism involved in this lack of insulin secretion is unclear.The level of vascular endothelial growth factor B(VEGF-B) is significantly increased in T2 D patients.The inactivation of VEGF-B could restore insulin sensitivity in db/db mice by reducing fatty acid accumulation.It is speculated that VEGF-B is related to pancreatic β-cell dysfunction and is an important factor affecting β-cell secretion of insulin.As an in vitro model of normal pancreatic β-cells,the MIN6 cell line can be used to analyze the mechanism of insulin secretion and related biological effects.AIM To study the role of VEGF-B in the insulin secretion signaling pathway in MIN6 cells and explore the effect of VEGF-B on blood glucose regulation.METHODS The MIN6 mouse pancreatic islet β-cell line was used as the model system.By administering exogenous VEGF-B protein or knocking down VEGF-B expression in MIN6 cells,we examined the effects of VEGF-B on insulin secretion,Ca2+ and cyclic adenosine monophosphate(cAMP) levels,and the insulin secretion signaling pathway.RESULTS Exogenous VEGF-B inhibited the secretion of insulin and simultaneously reduced the levels of Ca2+ and cAMP in MIN6 cells.Exogenous VEGF-B also reduced the expression of phospholipase C gamma 1(PLCγ1),phosphatidylinositol 3-kinase(PI3 K),serine/threonine kinase(AKT),and other proteins in the insulin secretion pathway.Upon knockdown of VEGF-B,MIN6 cells exhibited increased insulin secretion and Ca2+ and cAMP levels and upregulated expression of PLCγ1,PI3 K,AKT,and other proteins.CONCLUSION VEGF-B can regulate insulin secretion by modulating the levels of Ca2+ and cAMP.VEGF-B involvement in insulin secretion is related to the expression of PLCγ1,PI3 K,AKT,and other signaling proteins.These results provide theoretical support and an experimental basis for the study of VEGF-B in the pathogenesis of T2 D.
文摘Objective: To assess the clinical features, survival and prognostic factors of primary testicular diffuse large B-cell lymphoma (DLBCL). Methods: A retrospective study of 37 patients with primary testicular DLBCL was carried out from November 2003 to May 2012. Their clinical features, survival and prognostic factors were analyzed. Results: During a median follow-up period of 39.8 months (5.4-93.0 months), the median progression-free survival (PFS) was 26.2 months (95% CI:0-65 months) and the 3-year overall survival (OS) rate was 78.4%. Within the whole cohort, the factors significantly associated with a superior PFS were limited stage (stage Ⅰ/Ⅱ), lactate dehydrogenase (LDH) ≤245 U/L, international prognostic index (IPI) ≤1, primary tumor diameter 〈7.5 cm, and patients who had complete response (CR) and received doxoruhicin-contained chemotherapy (P〈0.05). There was a trend toward superior outcome for patients who received combined therapy (surgery/ chemotherapy/radiotherapy) (P=0.055). Patients who had CR, primary tumor diameter 〈7.5 cm and IPI score ≤1 were significantly associated with longer PFS at multivariate analysis. Conclusions: Primary testicular DLBCL had poorer survival. CR, primary tumor diameter and IPI were independent prognostic factors. The combined therapy of orchectomy, doxorubicin-contained chemotherapy and contralateral testicular radiotherapy (RT) seemed to improve survival.
文摘A human B cell line (3D5) that responds specifically to B cell growth factor (BCGF) hasbeen developed by a sequence of Staphylococcus aureus Cowen I activation,EB virus im-mortalization,and cloning.Proliferative response to PHA-stimulated T cell supernatant(PHA-T-Sup) and nonresponsiveness to rIL-2 stimulation were factors used to screen positivecells.Phenotype analysis with a flow cytometer indicated that:1) 3D5 is a B cell line:100% of the cells were positive for B1 marker and 59% were positive for sIg,while T3and Mo 1 were negative:2) 3D5 is an activated B cell line:both Tac and 4F2 markersof activated (but not of resting) B cells were 100% positive:3) 3D5 expresses high molecularweight BCGF (HMW-BCGF) receptor-associated epitope BA5.3D5 cells proliferated inresponse to cpBCGF stimulation in a dose-dependent manner.HMW-BCGF also induced3D5 cells to proliferate.Interestingly.no proliferation could be detected in the presenceof rIL-2,rIL-4,or rIFN-r.The data show that 3D5 cells are specifically BCGF-responsiveB cells.Using 3D5 cells as target,BCGF activity was detected in crude BCGF preparationsedimented by 85% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> and chromatographed in a DEAE-Sephadex A-25 column fromPHA-T-Sup.T24 cell supernatant with B cell differentiation factor (BCDF) activity couldnot induce 3D5 cells to differentiate into immunoglobulin-secreting cells.
基金Supported by the Outstanding Young Medical Personnel of Qingdao City
文摘AIM:To study the inhibition of nuclear factor kappa-B p65(NF-κB p65)antisense oligodeoxynucleotide(ASODN)on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2(TGF-β2).·M ETHODS:NF-κBp65ASODNand NF-κBp65missense oligodeoxynucleotide(MSODN)were designed and synthesized.Human lens epithelial cell line(HLE B-3)cells were prepared for study and divided into 7 groups.Control group was HLE B-3 cells cultured in dulbecco’s modified eagle medium(DMEM).T1,T2,and T3 group were HLE B-3 cells cultured in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A+T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M+T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h.The negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent(Hi Per Fect)for 24h.Cell morphology was observed at different time points using an inverted microscope.The expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction(RT-PCR),and the expression ofα-smooth muscle actin(α-SMA)protein was assayed with ELISA.·RESULTS:With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant(〈0.05).NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.NF-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.The difference between control group and A+T group was not statistically significant(〉0.05),but the difference among A+T group and other groups was statistically significant(〈0.05).·CONCLUSION:NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiation of HLE B-3.NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.
文摘The relation between the expression and activity of MMP-9 in C-reactive protein (CRP)-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B (NF-κB) was studied to investigate the possible role of CRP in plaque destabilization. Human THP-1 cells were incubated in the presence of CRP at 0 (control group), 25, 50 and 100 μg/mL (CRP groups) for 24 h. In PDTC (a specific NF-κB inhibitor) group, the cells were pre-treated with PDTC at 10 μmol/L and then with 100 μg/mL CRP. The conditioned media (CM) and human THP-1 cells in different groups were harvested. MMP-9 expression in CM and human THP-1 cells was measured by ELISA and Western blotting. MMP-9 activity was assessed by fluorogenic substrates. The expression of NF-κB inhibitor α (IκB-α) and NF-κB p65 was detected by Western blotting and ELISA respectively. The results showed that CRP increased the expression and activity of MMP-9 in a dose-dependent manner in the human THP-1 cells. Western blotting revealed that IiB-α expression was decreased in the cells with the concentrations of CRP and ELISA demonstrated that NF-κB p65 expression in the CRP-induced cells was increased. After pre-treatment of the cells with PDTC at 10 μmol/L, the decrease in IκB-α expression and the increase in NF-κB p65 expression in the CRP-induced cells were inhibited, and the expression and activity of MMP-9 were lowered too. It is concluded that increased expression and activity of MMP-9 in CRP-induced human THP-1 cells may be associated with activation of NF-κB. Down-regulation of the expression and activity of MMP-9 may be a new treatment alternative for plaque stabilization by inhibiting the NF-κB activation.
基金This projectwas supported byagrantfrom National Natu-ral Sciences Foundation of China (No. 39730 2 2 0 ) andWuhan Chenguang Program (No. 985 0 0 30 86)
文摘By study on the effect of anisodamine on lipopolysaccharide- induced expression of tissue factor(TF) in vascular endothelial cells(EC) ,the mechanism of anisodam ine antithrom bosis,as well as in the treatm ent of bacteraemic shock was investigated.Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion m ethod.TF activity was measured in the lysates of HUVEC by using a single step clotting assay.Specific m RNA expression was detected by Northern blotting.In order to evaluate a possible contribution of the nuclear factor (NF) -κB pathway on the effects observed,electrophoretic mobility shift assays(EMSA) were performed using nuclear extracts from HU VECs and NF- κB- binding oligonucleotides.The results showed that treatment of HUVEC with L PS resulted in a significant increase in TF activity.Anisodamine dose- dependently inhibited L PS- induced upregulation of TF.These effects was also confirm ed on the level of specific TF m RNA expression by Northern blotting.Furtherm ore,EMSA showed that anisodamine com pletely abolished L PS- induced NF-κB DNA binding activity in nuclear ex- tracts from HUVECs treated with L PS together with anisodamine.The results suggest thataniso- damine counteracts endothelial cell activation by inhibiting L PS- induced TF expression in these cells.Its interference with the NF- κB pathway might- at least in part- contribute to this effect. The ability of anisodamine to counteract L PS effect on endothelial cells m ight be one underlying m echanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.
基金supported by the Foundation of Stomatology Hospital,Xi'an Jiaotong University
文摘Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.
基金supported by grants from the National "973" Basic Research Program of China (No.2012CB944703)the National Key Technology Research and Development Program of China (No.2011BAI17B00)the Shandong Provincial Science and Technology Development Projects (No.2009GG10002008 and No.2011GSF12103)
文摘Objective: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1). Methods: A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni2+-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion: The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.