CAJ | 학술논문

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、72 h,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(α-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;流式细胞仪检测HSC凋亡率; Western blot、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72 h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.
목적 탐토대서골수간충질간세포(BMSC)여간성상세포(HSC)공배양체계중BMSC방분비간세포생장인자(HGF)대HSC조망적영향급기궤제. 방법 용반투막건립상하쌍층세포공배양체계,각조배양24、48、72 h,도치상차현미경관찰세포형태학변화;면역세포화학법관찰α-기동단백(α-SMA)표체정황;사갑기우담서염법검측Y-27632급PHA665752적최가간예농도;류식세포의검측HSC조망솔; Western blot、실시형광정량PCR검측간성상세포RhoAmRNA급단백표체정황;매련면역흡부법검측HGF급간세포생장인자격활인자(HGFA)농도.계량자료채용단인소방차분석,P＜ 0.05위차이유통계학의의.결과 수착시간적연장,HSC조망솔축점증고,실험b조조망솔최저,실험c조조망솔최고,균이72 h최위현저,각조재각시단조망솔차이구유통계학의의(P＜ 0.05);실험c조RhoA단백급mRNA표체량교기타각조명현하강,차이유통계학의의(P＜ 0.05);각조RhoA단백급mRNA표체량수시간적연장이축점증가;실험각조HGF농도수시간연장축점강저,실험b、c조교대조조증고,차이유통계학의의(P＜0.05);실험각조HGFA농도수시간연장축점증고,실험b조승고최위명현,차이유통계학의의(P＜0.01).결론 BMSC통과격활HGF병하조Rho통로촉진간성상세포조망.
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 AnnexinV-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.Results The apoptosis rates of HSCs 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 in a time-dependent manner,and the PHA665752 pretreated group showed significantly higher levels than any of the other groups at all time points examined (P ＜ 0.01).Conclusion BMSC promotes HSC apoptosis in a co-culture system by activating HGF and down-regulating the RhoA signaling pathway.