Experimental Investigation Into Modulation Of Fibrosis Within Area Of Glaucoma Surgery By 10-hydroxycamptothecin

Object:We cultured human Tenon's capsule fibroblast (HTFs) in vitro,investigated the effect of 10-hydroxycamptothecin on the proliferation and migration of human Tenon's capsule fibroblasts, and explored its possible mechanism with mitomycin C (MMC) contrast.Materials and Methods:1,The culture and identification of HTFs in vitro: We obtained fresh donor of normal Tenon capsule, cultured fibroblasts in vitro using tissue culture, and then obversed HTFs with light microscopy, immunofluorescence;2,The inhibitory effect of HCPT at different concentrations on HTFs was measured by methylthiazlyltetrazolium(MTT) assay, with mitomycin C (MMC) contrast;3,An FCM method was used to evaluate the cell cycles ;4,Scratch assays were used to evaluate the migration of HTFs;5,Trypan blue staining was used to evaluate the inhibitory of HCPT and MMC on HTFs whether caused by the cytotoxicity.6,Agarose gel electrophoresis was used to observe the apoptosis.7,Realtime PCR was used to detect the level of Smad7mRNA gene expression after HCPT,MMC on HTFs for 24h, exporing the possible mechanism.Results:1,HTFs were cultured successfully in vitro. The cells were long fusiformis , containing abundant cytoplasm and large nucleus. They grown strongly, showing swirling or feathery growth. HTFs were negatively stained with Cytokeratin and positively with Vimentin. Confirmed that the cultured cells were fibroblasts and not epithelial cells;.2,MTT assay showed that different concentrations of HCPT,MMC on HTFs for 24h, 48h, 72h ,the proliferation was lower than the control group, having significant differences; The inhibition rate of HCPT (0.031-0.125mg/l),MMC (0.0031-0.0125mg /l) on HTFs was not significantly higher, the inhibition rate of HCPT (0.25-4mg/l), MMC (0.025-0.4mg / l) on HTFs was significantly increased in a dose-dependent and time-dependent;IC50 of HCPT on HTFs for 24h,48h,72h was 2.24mg/l,0.76mg/l,0.39mg/l,IC50 of MMC on HTFs for 24h,48h,72h was 0.34mg/l,0.24mg/l,0.07mg/l, the inhibitory effect of MMC on the proliferation was 10 times than HCPT;3,An FCM method was used to evaluate the HCPT (0,0.25,1,4mg/l), MMC (0,0.025,0.1,0.4 mg/l) on HTFs cell cycle,showing that HCPT mainly impacted the G2 and S phase, MMC mainly blocked HTFs in G1 phase;4,Scratch assays were used to evaluate the migration of HTFs after different concentrations of HCPT,MMC for 24h,48h,72h.The results showed that the migration of HTFs was obviously inhibited in a dose-dependen but no significant correlation with the time;5,Trypan blue staining was used to detect the rate of living cells after HCPT (0,0.25,0.5,1,2,4mg/l), MMC (0,0.025,0.05,0.1,0.2,0.4mg/l) on HTFs for 24h,showing that there were no significant difference between HCPT group(or MMC group) and the control group;6,After 0.4mg/Lmmc,4mg/lHCPT on HFTs for 24h,the extracted DNA showed apoptosis-specific laddering by 1% agarose gel electrophoresis; 7,After 0.4mg/Lmmc,4mg/lHCPT on HFTs for 24h,Realtime PCR showed that the level of Smad7 mRNA expression was significantly increased.Conclusion:1,HCPT and MMC on cultured HTFs significantly inhibit the proliferation, this inhibition is concentration and time dependent. The inhibitory effect of the two drugs on HTFs is that MMC is 10 times than HCPT;2,HCPT mainly impacts the G2 and S phase, MMC mainly blocks HTFs in G1 phase;3,The suppressive effect of HCPT and MMC on HTFs proliferation has nothing to do with cytotoxicity induced by the two drugs.4,HCPT and MMC can induce the apotosis of HTFs;5,The mechanism of HCPT inhibiting the proliferation of HTFs may be: it inhibits the proliferation and migration of HTFs by increasing Smad7 mRNA expression to block TGF-βsignaling pathway .