四逆散含药血清对大鼠肝干细胞WB-F344增殖分化的影响

Effects of serum containing Sini Powder on proliferation and differentiation of rat's hepatic stem cells WB-F344

目的研究四逆散(柴胡,白芍,枳实,甘草)含药血清诱导大鼠肝干细胞WB-F344分化的作用。方法 SD大鼠灌胃四逆散,取其含药血清处理WB-F344细胞,应用CCK-8方法检测细胞活性,Western blot法检测甲胎蛋白(AFP)、白蛋白(ALB)、细胞色素P450 3A(CYP 3A)蛋白表达,PCR检测AFP、ALB的mRNA水平,PAS法检测糖原合成功能,尿素氮检测试剂盒观察尿素氮合成功能,Mito Tracker Red染色法检测线粒体数目,Clark氧电极检测细胞耗氧量,荧光素酶法测定细胞内ATP水平,JC-1染色检测线粒体膜电位。结果含药血清对实验细胞无毒性作用,10%含药血清显示出较好的促进细胞增殖的效果;含药血清作用后,AFP的蛋白及基因水平有降低趋势,ALB的蛋白及基因水平有升高趋势,糖原合成功能、尿素氮合成功能提高,CYP 3A蛋白水平有一定增加。同时,线粒体数目增加,细胞呼吸功能、ATP水平、线粒体膜电位都有一定程度增加。结论四逆散含药血清可促进肝干细胞增殖、诱导其分化,可能为促进肝损伤修复的机制之一。

AIM To study the effects of serum containing Sini Powder（Bupleuri Radix,Paeoniae Radix alba,Aurantii Fructus immaturus,Glycyrrhizae Radix et Rhizoma） on prolification and differentiation of rat＇s hepatic stem cells WB-F344 and to explore their mechanism.METHODS Serums containing test drug collected from SD rats receiving Sini Powder by purfusion were co-cultured with WB-F344 cells.The activity of cells was detected by CCK-8 assay.The expressions of AFP,ALB and cytochrom P450 3A（CYP 3A） were determined by Western blot.The changes of mRNA level for AFP and ALB were examined by RT-PCR.Functional properties of mature hepatocytes were measured with PAS and Urea Nitrogen Assay Kit method while CYP 3A protein was assessed by Western-blot.The mitochondrial number,oxygen consumption rate,ATP content,mitochondrial membrane potential（MMP） were investigated by using Mito Tracker Red,Clark oxygen electrode,luciferin-luciferase method,JC-1,respectively.RESULTS Different concentrations of serums containing test drug showed no toxic effects on WB-F344 cells with 10% concentration significantly promoting proliferation of WB-F344 cells.The levels of AFP protein and gene expression both decreased but ALB trended to a up-regulation.WB-F344 cell functions concerning glycogen synthesis,urea nitrogen production,and CYP 3A protein and mitochondrial number and function（respiratory function,ATP content,MMP） all increased after incubating with serum containing the drug tested.CONCLUSION The serum containing Sini Powder can promote hepatic stem cells＇ proliferation and induce their differentiation.It may be one of the mechanisms for the repair of liver injury.