仿生电刺激在体外促进人骨髓间充质干细胞株向肝细胞分化的研究

摘要

目的 探讨仿生电刺激体外诱导人骨髓间充质干细胞株(UE7T-13)向肝细胞分化的作用.方法 采用静电纺丝及氧化聚合法制备聚吡咯/PLGA导电膜,并在其表面培养UE7T-13细胞株.实验按是否给予诱导因子及施加电刺激分为四组:电刺激+诱导因子组、电刺激组、诱导因子组以及空白对照组.诱导因子包括HGF、EGF、FGF及OSM等.电刺激为每日给予频率5Hz、脉宽5 ms、电压100 mV(电流强度100 μA)的仿生电刺激2h.每天常规收集培养液,检测其中白蛋白分泌、尿素合成、细胞色素P450活性.MTT法检测细胞增殖能力,细胞免疫荧光方法检测各组干细胞中白蛋白和甲胎蛋白的表达.结果 电刺激+诱导因子组及诱导因子组UE7T-13细胞均成功诱导出类肝样细胞.与单纯诱导因子组比较,电刺激+诱导因子组UE7T-13更早向肝细胞转化,其肝细胞特异功能表达及表面特异蛋白分子表达更强.UE7T-13细胞在有电刺激存在时增殖能力明显提高.结论 仿生电刺激可促进体外UE7T-13细胞增殖及其肝向分化,使其更早出现肝细胞特异性分子表达,并提高分化效率.%Objetive To explore the effects of biomimetic electrical stimulation on inducing differentiation of human bone marrow mesenchymal stem cells (UE7T-13) into hepatocytes in vitro.Methods The polypyrrole/PLGA conductive nanofiber scaffold was designed via electrospinning and oxidative polymerization,on which the UE7T-13 cells were cultured.The experiment were divided in to four groups according to supply of growth factors and electrical stimulation,that is:growth factor and electrical stimulation group (GF-ES group),electrical stimulation group (ES group),growth factor group (GF group),and control group (Ctr group).Suprathreshold square biphasic pulses stimulation (5 ms duration,5 Hz,100 μA)were given in ES group lasting for 2 h each day.And FGF,HGF,OSM,EGF were given in GF group.The cell proliferation was measured by MTT assay.The albumin secretion and urea synthesis were detected by ELISA assay,the cytochrome P450 activity was detected by fluorescent quantitative detection kit,and the expressions of albumin and AFP of MSCs in each group were detected by immunofluorescence.Results Hepatocyte-like cells were induced in both GF-ES group and GF group,and cells in the GF-ES group showed higher expression of albumin and earlier expression of AFP than those in the GF group.The urea synthesis,albumin secretion and CYP1A2 expression in GF-ES group were also higher than GF group.In addition,the UE7T-13 cells showed stronger proliferation ability under the condition of electrical stimulation.Conclusion Biomimetic electrical stimulation could promote both proliferation and hepatic differentiation of UE7T-13 cells with earlier exhibition of hepatocyte-specific marker proteins,so as to enhance the differentiation efficiency.