The Study Of The Effect Of Tanshinone â…¡A On Fibroblasts Of Rabbit Benign Stricture Bile Duct

Objective:This study is aimed to examine the effects of TanshinoneⅡA on the fibroblasts of rabbit benign stricture bile duct proliferation,Secretion of collagen typeⅠand the expression of matrix metalloproteinases-9.To explore the mechanism of Chinese medicine treatment benign biliary strictures and to provide a theoretical basis of clinical application of Chinese medicine for the treatment of benign bile duct stricture.Methods:Establish the benign bile duct stricture model by jawing extrohepatic bile ductin rabbit,and 72h later,catch the bile duct tissues in that rabbit.FB cells were isolated by collagenase digestion and tissue culture,Observed fibroblasts morphology under inverted microscope;Passages 3-5 cells were used for the experiments.They were divided into control solution and tanshinoneⅡA group. Detection the cells proliferation and the influence of different concentrations (0.1ug/ml,0.5ug/ml,2.5ug/ml,10ug/ml and 25ug/ml) tanshinoneⅡA on FB at 1st day, 2nd day,3 days by MTT and the levels of collagen typeⅠquantitatively by ELISA method;Determination of the mRNA expression of MMP-9 by RT-PCR method.Results:(1) It's obvious that hyperemia,edema and clamp traces after clamped the common bile duct.When re-open after 72h,we found the bile duct organizations clearly extravagated blood swelling and the expansion of proximal bile duct.Bile duct and surrounding tissue with mild adhesions,however,very loose,easy to separate;And Mild cholestatic liver performance can be seen.(2) Cultured cells showed typical fibroblast morphology,arranged spindle,polygonal or irregular in shape.Cell cytoplasm is characterized by transparency,it is very thin;nucleus large, oval-shaped,color,light,usually with 2 to 3 nuclear.(3) MTT assay of tanshinoneⅡA in rabbit bile duct fibroblasts affected were as follows:Compared with control group, when the concentration of tanshinonⅡA is 0.1μg/m,0.5μg/m,2.5μg/ml,it's had no significant effect on rabbit bile duct fibroblasts(P＞0.05);when the concentration of tanshinoneⅡA is 10μg/ml,the 72-hour show that inhibit fibroblasts proliferation of rabbit bile duct cells(P＜0.05),when the concentration of tanshinoneⅡA is 25μg/ml, the 24-hour show that inhibit fibroblasts proliferation of rabbit bile duct cells (P＜0.05),and as the extension of time and the increase in drug concentration,the role of inhibition enhanced;(4) Elisa assay of tanshinoneⅡA effect on rabbit bile duct fibroblasts secretion of collagen typeⅠfound:TanshinoneⅡA group compared with the control group,fibroblasts secrete collagen typeⅠwere significantly reduced (P＜0.01);(5) After tanshinoneⅡA solution work on 48h,the expression of MMP-9mRNA increased significantly(P＜0.05).Conclusion:(1) Clamp method in rabbit bile duct fibroblasts cell culture method is a good access to bile duct fibroblasts method.Enzymatic digestion - tissue culture is a better method of cell culture.(2) TanshinoneⅡA,monomer composition of traditional Chinese medicine Danshen,effectively inhibit benign stricture of bile duct fibroblasts proliferation and collagen typeⅠsecretion,and tanshinoneⅡA can induce the expression of MMP-9mRNA promote collagen degradation,thereby inhibiting the formation of bile duct scar.