【作者】 彭雪彬；

【导师】 陈红；

【作者基本信息】 兰州大学 ， 内科学， 2013， 硕士

【摘要】 目的探讨在体外共培养条件下,人脐带间充质干细胞对肝星状细胞增殖的调控机制。方法采用Transwell间接共培养体系,实验组上层接种人脐带间充质干细胞(UCMSCs),下层接种肝星状细胞(HSCs-LX-2);对照组仅肝星状细胞单独培养。MTT法测肝星状细胞增殖抑制率；HOECHST染色法,直接观察肝星状细胞增殖抑制情况；流式分析技术检测各组肝星状细胞凋亡情况；ELISA法检测各实验组细胞上清液中TGF-β1蛋白的表达；荧光实时定量PCR检测各组肝星状细胞中TGF-β1、Smad3、Smad7mRNA的表达；Western blot法检测各组肝星状细胞TGF-β1、Smad3、Smad7蛋白的表达。结果与对照组比较,实验组共培养24、48h,随着时间延长肝星状细胞增殖抑制率明显升高(P<0.05)；HOECHST染色可见实验共培养组肝星状细胞凋亡增多,且48h较24h更明显(P<0.05)；流式细胞分析示共培养组肝星状细胞凋亡多于对照组,且48h较24h更明显(P<0.05)；上清液中,共培养组TGF-β1较对照组少,随着时间增加,差异更明显(P<0.05)。共培养48h实验组TGF-β1及Smad3mRNA表达低于对照组,而Smad7mRNA表达高于对照组(P<0.05)。共培养24h、48hTGF-β1、Smad3蛋白的表达均低于对照组,Smad7蛋白表达高于对照组(P<0.05)。结论人脐带间充质干细胞能够抑制肝星状细胞的增殖,促进其凋亡,其机制可能是通过下调TGF-β1、Smad3,上调Smad7蛋白表达而实现的。更多还原

【Abstract】 Objective To explore the mechanism of human umbilical cord mesenchymal stem cells (UCMSCs) to regulate the proliferation of hepatic stellate cells (HSCs-LX2) under co-culture in vitro.Methods Human UCMSCs and HSCs in the experimental group were cultured in the plastic culture plate(6holes) to establish the upper and lower double-cell co-culture system. HSCs were cultured alone as negative control group. Cell proliferation and apoptosis were determined by MTT assay and flow cytometry, respectively. Cell supernatants were harvested to determine the concentration of transforming growth factor betal (TGF-β1) by enzyme-linked immunosorbent assay (ELISA). The TGF-β1, Smad3and Smad7mRNA expression in HSC was determined by reverse transcription-polymerase chain reaction (RT-PCR) and the level of TGF-β1, Smad3and Smad7protein by Western blot..Results HSCs co-cultured with UCMSCs significantly inhibited HSC proliferation compared with the negative control group at24,48hours(P<0.05); Flow cytometry showed that HSCs apoptosis of co-culture group was increased compared with negative control group, and48h is more obvious (P<0.05). The concentrations of TGF-β1in co-culture supernatants in the experimental group was significantly lower than those in the supernatants of HSCs cultured alone, and48h is more obvious (P<0.05). After HSCs co-cultured with BMSCs for48hours, the expression of TGF-β1, Smad3mRNA and protein was reduced and the expression of Smad7mRNA and protein was increased compared with negative control group (P<0.05).Conclusion UCMSCs inhibited the proliferation of HSCs, possibly through inhibiting the TGF-β1and Smad3expression, increasing Smad7protein expression.更多还原