珠子参皂苷诱导骨髓干细胞分化为肝样细胞的体外实验研究

The study of saponins from Panacis majoris on the differentiation of bone marrow stem cells into hepatocytes

目的:观察珠子参皂苷对骨髓干细胞(bone marrow stem cells,BMSCs)分化为肝细胞的影响。方法:在无菌条件下快速处死SD大鼠,收集骨髓细胞,快速悬浮于预冷的NH4Cl低渗盐溶液中裂解红细胞,离心去上清,收集沉淀的细胞,D-HankS充分洗涤后,用含10%的低糖型DMEM培养基进行扩增培养;取生长状态良好的第四代细胞,随机选择空白组、HGF组(20ng/ml)、珠子参皂苷(50、100和200μg/ml)组,分别与第7天和第14天后观察细胞形态、检测细胞合成糖原,摄取LDL及分泌尿素的能力,进行HGF、c-MET、AFP、Albumin、CK18基因表达和AFP、Albumin、CK18蛋白表达分析。结果:珠子参皂苷和HGF可诱导BMSCs分化为肝细胞,促进其合成糖原、摄取LDL及分泌尿素的能力;可显著增加肝细胞特异性HGF、c-MET、AFP、Albumin、CK18基因表达和AFP、Albumin、CK18蛋白表达。结论:珠子参皂苷体外能促进BMSCs分化为肝细胞,本研究为后续体内进行BMSCs移植和珠子参皂苷联用治疗终末期肝病(ESLD)提供了理论基础和实验依据。

Objective： To observe the influence of saponins from Panacis majoris on the differentiation of hepatocyte from bone marrow stem cells（ BMSCs）. Methods： Under sterile conditions,Sprague Dawley rats were sacrificed,bone marrow cells were collected,suspended in the NH4 Cl hypotonic salt solution（ erythroeyte lysis bufferr） for erythrocyte lysis,and centrifuged to collect the cells for cultivating. Low glueose DMEM（ LG-DMEM）,supplemented with10% fetal bovine serum（ FBS） was applied. When the culture reached 80% confluence,it was preincubated with collagenase for l-2 hours,cells were trysinized and aserial subcultivation began. The fourth generation cells with good growth state were randomly control,HGF（ 20 ng / ml）,and the saponins from Panacis majoris（ 50,100 and 200μg / ml）,the cell morphology was observed in seventh days and fourteenth days,respectively,analyzed the cell ability of synthesizing glycogen,uptaking LDL and secreting urea,The mRNA expressions of HGF,c-MET,AFP,Albumin and CK18 were detected by real-time polymerase chain reaction; The protein expressions of AFP,Albumin and CK18 in the cells were detected by western blotting. Results： Saponins from Panacis majoris（ 50,100 and200μg / ml） and HGF（ 20 ng / ml） induced the BMSCs to differentiate into hepatocytes,significantly increased the mRNA expressions of HGF,c-MET,AFP,Albumin and CK18,and the protein expressions of AFP,Albumin and CK18. Conclusion： Our present study indicated that saponins from Panacis majoris could effectively promote BMSCs differentiation into hepatocytes in vitro,which provided a theoretical basis for the following BMSCs transplantation in vivo and the theoretical and experimental basis for the treatment of ESLD with combined application.