大鼠骨髓间充质干细胞促进肝星状细胞凋亡的机制研究

Mechanism of bone marrow-derived mesenchymal stem cell-promoted apoptosis of hepatic stellate cells

目的探讨大鼠骨髓间充质干细胞（BMSC）与肝星状细胞（HSC）共培养体系中BMSC旁分泌肝细胞生长因子（HGF）对HSC凋亡的影响及其机制。方法用半透膜建立上下双层细胞共培养体系，各组培养24、48、72h，倒置相差显微镜观察细胞形态学变化，免疫细胞化学法观察α-肌动蛋白（仅-SMA）表达情况；四甲基偶氮唑盐法检测Y-27632及PHA665752的最佳干预浓度l流式细胞仪检测HSC凋亡率lWesternNot、实时荧光定量PCR检测肝星状细胞RhoAmRNA及蛋白表达情况；酶联免疫吸附法检测HGF及肝细胞生长因子激活因子（HGFA）浓度。计量资料采用单因素方差分析，P〈0．05为差异有统计学意义。结果随着时间的延长，HSC凋亡率逐渐增高，实验b组凋亡率最低，实验c组凋亡率最高，均以72h最为显著，各组在各时段凋亡率差异具有统计学意义（P〈0．05）；实验C组RhoA蛋白及mRNA表达量较其他各组明显下降，差异有统计学意义（P〈0．05）；各组RhoA蛋白及mRNA表达量随时间的延长而逐渐增加；实验各组HGF浓度随时间延长逐渐降低，实验b、c组较对照组增高，差异有统计学意义（P〈0．05）；实验各组HGFA浓度随时间延长逐渐增高，实验b组升高最为明显，差异有统计学意义（P〈0．01）。结论BMSC通过激活HGF并下调Rho通路促进肝星状细胞凋亡。

Objective To explore the role of the Rho pathway in the hepatocyte growth factor （HGF） paracrine signal-mediated bone marrow-derived mesenchymal stem cell （BMSC） promotion of apoptosis of hepatic stellate cells （HSCs）. Methods A BMSC-HSC co-culture system was established using plates with transwell inserts. Dynamic changes in response to pretreatment with the c-met blocker PHA665752 and the Rho pathway inhibitor Y-27632 were observed under an inverted phase contrast microscope at 24, 48 and 72 h of culture. Optimal intervention concentrations of Y-27632 and PHA665752 were determined by MTT assay. Expression of alpha-smooth muscle actin in HSCs was evaluated by immunohistochemistry, and the apoptosis rate of HSCs was measured by Annexin- V-FITC/propidium iodide. RhoA protein and mRNA levels were measured by western blot and quantitative real-time PCR respectively. Concentrations of HGF and hepatocyte growth factor activator （HGFA） were quantified by enzyme-linked immunosorbent assay. Between-group differences were evaluated by one-way ANOVA with P 〈 0.05 indicating significance. ResuRs The apoptosis rates ofHSCs gradually and steadily increased in a time-dependent manner. The apoptosis rate of the PHA665752 pretreated group was lowest and that of the Y-27632 pretreated group was highest, with the most robust difference occurring at the 72 h time point （P 〈 0.05）. The mRNA and protein expression levels of RhoA decreased in a time-dependent manner in the Y-27632 pretreated group （all time points, P 〈 0.05） but the expression levels increased in a time-dependent manner in the PHA665752 pretreated group （all time points, P 〈 0.05）. For both the PHA665752 and the Y-27632 pretreated groups, the concentration of HGF decreased in a time-dependent manner, but the concentrations in both remained significantly higher than that in the control group at all time points examined （P 〈 0.05）. The concentration of HGFA increased ha a time-dependent manner, and the PHA665752 pretreated group showed significantly higher levels than any of the other groups at aU time points examined （P 〈 0.01）. Conclusion BMSC promotes HSC apoptosis in a co-culture system by activating HGF and down-remalatine the RhoA signaling pathwav.