剔毒护肝方及其拆方对大鼠骨髓干细胞增殖分化的影响

Influence of Tidu Hugan recipe and separated recipes on the proliferation and differentiation of rat bone marrow stem cell

目的:观察剔毒护肝方及其拆方含药血清对大鼠骨髓干细胞增殖作用和向肝系分化的影响。方法:40只Wistar大鼠被随机分为剔毒护肝方组、黄芪组、莪术组、叶下珠组、生理盐水对照组、肝再生血清组,制作药物血清;使用密度梯度离心和贴壁筛选相结合,分离骨髓干细胞;通过噻唑氮蓝(MTT)比色法检测含药血清处理48小时时骨髓干细胞的增殖率;采用免疫组化法检测骨髓干细胞肝系标志(AFP、白蛋白)表达情况。结果:经各组血清培养48小时后,剔毒护肝方组、黄芪组和肝再生血清组对大鼠骨髓干细胞增殖率分别为10.4%、14.3%和26.5%;剔毒护肝方组、黄芪组、肝再生血清组,AFP和白蛋白表达情况强于其他组;剔毒护肝方组、黄芪组、肝再生血清组可见糖原阳性表达。结论:剔毒护肝方组、黄芪组含药血清对骨髓干细胞有增殖作用,可诱导骨髓干细胞横向分化为肝实质细胞。剔毒护肝方中发挥主要作用的药物可能是黄芪,但剔毒护肝方的作用强于黄芪组,说明组成剔毒护肝方的三味药不是简单的叠加。

Objective： To explore whether bone marrow stem cells （BMSCs） from rat can be induced to differentiate into hepatocytes by Tidu Hugan recipe and separated recipes （astragalus, zedoary and phyllanthus）. Methods： Forty Wistar male rats were randomly divided into 7 groups ： Tidu Hugan recipe group, astragalus group, zedoary group, phyllanthus group, sodium chloride of normal control group, hepatectomized rat serum group, isolation of BMSCs group. The mononuclear cells of the marrow was obtained by gradient centrifugation in the Percoll （density 1. 077g/L） and sticking on flask. MTT colorimetric assay were used to observe the effect on the growth and proliferation in the cell treated with every group. The expression of alpha-fetoprotein （AFP） and Albumin （Alb） were examinated by immuno-histochemical method after treated with serum containing Tidu Hugan recipe and its different combined herbs, and hepatectomized rat serum. The function of hepatocyte was examinated the glycogen production. Result： Both zedoary and phyllanthus inhibit the proliferation of BMSCs, but Tidu Hugan recipe, astragalus and hepatectomized rat serum promote the proliferation of BMSCs, and the growth rate was 10.4%, 14. 3%, 26. 5% respectively. Tidu Hugan recipe, astragalus and hepatectomized rat serum were positive in expressing Alb and AFP in immunohistochemistry, radio-immunity quantitation in AFP, Tidu Hugan recipe was obviously higher than others, and radio-immunity quantitation in Alb had no obvious difference in every group. Tidu Hugan recipe, astragalus and hepatectomized rat serum were positive in glycogen detection. Conclusion： Tidu Hugan recipe and astragalus promote the proliferation of BMSCs, and they can induce the rat BMSCs differentiate into the hepatocyte through the expression of AFP, Alb and the glycogen production. In Tidu Hugan recipe produce a marked effect maybe astragalus, but Tidu Hugan recipe was more effective than astragalus. So in Tidu Hugan recipe the other two herbs also produce some effect, the specific mechanism need deeper study.