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Objective To study the effect and mechanisms of chloride channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) on thansforming growth factor β1 (TGF-β1) induced human conjunctival fibroblasts (HConF) fibrosis.Methods Cell counting kit (CCK-8) was used to screen out the optimal TGF-β1 treatment time and the optimal NPPB concentration.The cells were divided into control group,TGF-β1 treatment group and TGF-β1+NPPB group.Cell proliferation and cell cycle were detected by CCK-8 and flow cytometer,respectively.Cell migration ability were observed by scratch and transwell migration assays.Western blot and Real time-PCR were used to detect the expression of collagen Ⅰ (COL-Ⅰ),fibronectin (FN) and α-smooth muscle actin (α-SMA).The phosphorylation level of PI3K and Akt were measured by Western blot.Results TGF-β1 promotes cell proliferation in a time-dependent manner.There was no statistically significant difference in A values between 48 hours and 72 hours after TGF-β1 treatment (P =0.064).Forty-eight hours was selected as the most appropriate time for TGF-β1 treatment.NPPB inhibited HConF cell proliferation in a concentration-dependent manner.Compared with the control group,the proliferation A values of cells in the 50 mol/L and 100 mol/L NPPB groups were significantly reduced (P =0.020,0.000),and 100 mol/L was selected as the optimal concentration of NPPB.The cell proliferation A value,migration area and migration cell number of TGF-β1 +NPPB group were significantly lower than those of TGF-β1 treatment group (all at P＜0.05).Compared with the control group and TGF-β1 +NPPB group,the proportion of G1 phase cells in the TGF-β1 treatment group was reduced,and the proportion of cells in the S phase and G2/M phase were increased,with statistically significant differences between them (all at P ＜ 0.05).The protein and mRNA expression of α-SMA,COL-Ⅰ and FN in the TGF-β1 treatment group were higher than those in the control group and TGF-β1+NPPB group,with statistically significant differences between them(all at P＜0.05);the ratios of p-PI3K/PI3K and p-Akt/Akt in the TGF-β1 treatment group were significantly higher than those in the control group and TGF-β1 +NPPB group,with statistically significant differences between them (all at P＜0.05).Conclusions NPPB may inhibit TGF-β1 induced HConF fibrosis process by inhibiting phosphorylation of PI3K and Akt.
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Objective To analyze the influence of dexamethasone in the hypotonic-induced volume-sensitive chloride currents in human trabecular meshwork cells,and to investigate the possible mechanism of volume-sensitive chloride channels(VACC)in the glucocorticoid-induced glaucoma(GIG)cases.Methods The human trabecular meshwork cells were seeded in 35 mm diameter plastic petri dishes,so that they could grow in monolayer.The cultured cells were divided into normal cell culture medium group and dexamethasone 1 d,3 d,7 d groups.The chlorine current density values of the cells in four groups were recorded respectively by the whole-cell patch-clamp technique. The differences among groups were compared.Results In normal group,after hypotonic stimulation,under+100 and-100 mV voltage clamp,the outward and inward current density values of the trabecular cells were (19.94±0.87) and (-6.53±0.41)pA/pF.In dexamethasone 1 d,3 d,and 7 d groups,under the same condition,the outward and inward current density values of the trabecular cells were (19.39 ± 1.40)and (-6.42 ± 0.28)pA/pF, (17.97±2.35)and (-5.82±0.94)pA/pF,(17.16±1.16)and (-5.65±0.43)pA/pF.The trabecular cells cultured with dexamethasone for 1 d had lower outward and inward current density values under hypotonic stimulation compared with normal group,but there was no significant difference (P>0.05).The trabecular cells cultured with dexamethasone for 3 d and 7 d,when compared with normal group,had significantly lower outward and inward current density values under hypotonic stimulation (P<0.05 ). Conclusion Dexamethasone could reduce the volume-sensitive chloride current in trabecular meshwork cells,which would affect trabecular meshwork cell volume adjustment.This would possibly cause the increase of the aqueous humor outflow resistance among GIG cases.
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Objective To evaluate the effect of hepatocyte growth factor(HGF) on transvascular metastasis of sarcoma cells.Methods The models of intravascular and extravascular migration of tumor cells in vitro were used to observe transvascular metastasis of sarcoma cells(HT1080).Results HT1080 migrated through basement membrane into blood vessels,and migrated through a gap between adjacent endothelial cells into extracellular matrix.The greater number of transmigrated HT1080 treated with HGF was observed(P
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0.05). Phagocytic index of RPE cells was (28.7?1.9)% in the presence of 10 ?mol?L~ -1 DIDS,10 ?mol?L~ -1 DIDS significantly inhibited the nonspecific phagocytic process of human RPE cells(P
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Objective:To investigate if chloride channel ClC-2 could be phosphorylated by mitogen activated protein kinase(MAPK) for further study of its regulation mechanism in proliferation and differentiation of cells.Methods:The coding sequence containing the cytosolic C-terminus of ClC-2 was amplified from pSPORT1/ClC-2 plasmid,including rabbit ClC-2 cDNA, by polymerase chain reaction(PCR),the fragment was cloned into pGEX-4T-1 plasmid for the construction of GST-tagged fusion protein expressing vector, pGEX-4T-1/ClC-2CT.After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E.coli BL21. The expression of GST-tagged fusion protein was induced with IPTG and purified with Gluthathion Sepharose 4B affinity chromatography. Then the phosphorylation of ClC-2 by MAPK was examined by using phosphorylation assays in vitro.Results:The construction of pGEX-4T-1/ClC-2CT recombinant vector was proved by enzyme digestion and sequencing. The purified fusion protein GST/ClC-2CT could be phosphorylated by MAPK, however the GST could not.Conclusion:Chloride channel ClC-2 can be phosphorylated by MAPK.
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AIM: To evaluate the effect of endothelial Rho and Rho kinase in extravasation migration of sarcoma cell. METHODS: We used an in vitro model of sarcoma cell transmigration across a monolayer of HUVEC cultured on collagen gel to observe extravasation migration of sarcoma cells and calculated the electrical resistance of HUVEC monolayer in extravasation migration of sarcoma cells. RESULTS: Sarcoma cells migrated through endothelial cells into collagen gel, the electrical resistance of a HUVEC monolayer reduced in extravasation migration of sarcoma cells.Endothelial Rho inhibitor(C3 transferase) and Rho kinase inhibitor(Y-27632) inhibited extravasation migration of sarcoma cells and inhibited reduction of electrical resistance of a HUVEC monolayer in extravasation migration of sarcoma cells. CONCLUSION: Endothelial Rho and Rho kinase regulates sarcoma cell transendothelial migration through modification of endothelial cytoskeleton.
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AIM: To evaluate the effect of tissue factor on intravascular migration of tumor cells.METHODS: Expression of tissue factor in tumor cells (HT1080) was analyzed by flow cytometry and immunofluorescence staining. An in vitro model was used to observe intravascular migration of tumor cells. RESULTS: High expression of tissue factor was observed in tumor cells (HT1080). The antibody for tissue factor inhibited intravascular migration of tumor cells. CONCLUSION: Tissue factor stimulated tumor metastasis through promoting intravascular migration of tumor cells.
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AIM: To evaluate the effect of endothelial myosin light chain kinase on extravasation migration of sarcoma cell. METHODS: An in vitro model of sarcoma cell transmigration across a monolayer of HUVEC cultured on collagen gel was used to observe extravasation migration of sarcoma cell and calculated the electrical resistance of HUVEC monolayer in extravasation migration of sarcoma cell. RESULTS: Sarcoma cell migrated through a gap between adjacent endothelial cells into collagen gel. The electrical resistance of a HUVEC monolayer reduced in extravasation migration of sarcoma cell.Endothelial myosin light chain kinase inhibitor(ML-7) inhibited extravasation migration of sarcoma cell and inhibited reduction of electrical resistance of a HUVEC monolayer in extravasation migration of sarcoma cell in a dose-dependent manner. CONCLUSION: Endothelial myosin light chain kinase regulates sarcoma cell transendothelial migration by phosphorylation of myosin light chain.