Effects Of Ginkgo Biloba Extract On Cell Proliferation In Human Tenon’s Capsule Fibroblasts Stimulated By TGF-β1and Its Potential Mechanism

Objective:To investigate the effects of Ginkgo biloba extract on cellproliferation in cultured human Tenon’s capsule fibroblasts and its potentialmechanism.Methods:1.Human Tenon’s capsule fibroblasts culture andidentification:Taken human tenon’s capule tissues from glaucoma filteringsurgery and cultured fibroblasts with tissue explants technique invitro.Fibroblasts were subclutured in5%CO2,37℃condition with DMEMcontained10%fetal bovine serum.Antibody of Vimentin and Keratin wereused in immunocytochemistry to indentify the cultured cells.2. Evaluate GBE on the proliferation of HTCFs induced by TGF-β1with MTT assay:The cultured cells were divided into6groups:controlgroup,TGF-β1(2ng/ml) group、TGF-β1(2ng/ml)+GBE（25、50、100、200mg/L） groups， proliferation of fibroblasts were detected by3-(4,5-dimethyl-2thiazol)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay after24、48、72hours in culture.3. Flow cytometry tested cell cycle:The cells were divided into 3groups(n=3),control groups、TGF-β1(2ng/ml) group and TGF-β1(2ng/ml)+GBE（200mg/L）group,cell cycle were tested after72hours inculture.4.Immunocytochemistry detected the expression of α-smoothmuscle-actin(α-SMA)：The cells were divided into3groups(n=3),controlgroups、TGF-β1(2ng/ml) group and TGF-β1(2ng/ml)+GBE（200mg/L）group，the expression of α-SMA were detected after72hours in culture.5. RT-PCR measured mRNA level of connective tissue growthfactor(CTGF): The cells were divided into3groups(n=3),control groups、TGF-β1(2ng/ml) group and TGF-β1(2ng/ml)+GBE（200mg/L）group,after72hours in culture,the level of CTGFmRNA were measured by RT-PCR.Results:1.The cells cultured in vitro proliferated actively,immune-histochemical staining of vimentin were positive and keratin were neg-ative.2. TGF-β1markedly increased the cell proliferation,GBE effectivelyinhibited the proliferation，and showed a concentration-time dependentway.3.TGF-β1promote HTCFs from G0/G1phase to S phase,the cells of Sphase significantly reduced after GBE treated(p<0.05).4.TGF-β1observably raised the expression of α-SMA， GBEsignificantly inhibited the effect of TGF-β1（p<0.05）.5.TGF-β1significantly raised the expression of CTGFmRNA，GBE markedly inhibited the effect of TGF-β1（p<0.05）.Conclusions：1.HTCFs is easy to culture in vitro，the character of thepassage cells is steady.The HTCFs is a good carrier in studying theantiscaring of flitering bleb.2.Ginkgo biloba extract can inhibit HTCFs proliferation,and itspotential mechanisms are that prevent cells enter S-phase，down-regulatethe TGF-β1induced α-SMA and CTGFmRNA overexpression.