骨髓间充质干细胞调控肝星状细胞RhoA、P27的表达  

Bone marrow mesenchymal stem cells modulate the expression of RhoA and P27 in hepatic stellate cells

作　　者：苏思标[1] 姜海行[1] 王东旭[1] 覃山羽[1] 梁梓宇[1] 

机构地区：[1]广西医科大学第一附属医院消化内科,广西壮族自治区南宁市530021

出　　处：《世界华人消化杂志》2009年第32期3283-3291,共9页World Chinese Journal of Digestology

基　　金：广西自然科学基金资助项目;No.0640133;No.0897008~~

摘　　要：目的:观察体外大鼠骨髓间充质干细胞(MSCs)对肝星状细胞(HSCs)RhoA信号因子及其细胞周期调控因子P27表达的影响,探讨MSCs调控HSCs细胞周期G1/S转换机制.方法:贴壁筛选法培养、纯化SD大鼠MSCs,传代至第4代使用;大鼠肝星状细胞(HSC-T6)系及纤维原细胞系冻融后传代使用.应用6孔塑料细胞培养盒,每孔使用半透膜(transwell insert)建立上下双层细胞共培养体系,常规培养.实验分3组:空白对照组、阴性对照组、MSCs实验组.用WST-8法对HSCs增殖率进行测定;流式细胞仪检测细胞周期;RT-PCR、Western blot检测MSCs与HSCs共培养后HSCs内RhoA,P27mRNA和蛋白的表达.结果:(1)HSCs与MSCs共培养24h后,HSCs表现明显增殖抑制(P<0.01),且呈现时间依赖性.MSCs实验组与空白对照组、阴性对照组比较均有显著性差异(均P<0.01).(2)共培养12h后,MSCs可阻滞HSCs由G0/G1期向S期转换,使G0/G1期细胞增多,S期细胞减少,与空白对照组、阴性对照组比较差异均有统计学意义(P<0.01).(3)共培养12h后,MSCs实验组RhoA mRNA表达与空白对照组、阴性对照组比较均有统计学差异(P<0.05,P<0.01),随时间的延长呈递减趋势;共培养后,MSCs实验组P27 mRNA表达与空白对照组、阴性对照组比较均无统计学差异.(4)共培养12h后,MSCs实验组RhoA蛋白表达与空白对照组、阴性对照组比较均有统计学差异(均P<0.01),随时间的延长呈递减趋势;共培养12h后,MSCs实验组P27蛋白与空白对照组、阴性对照组比较均有统计学差异(均P<0.01),随时间的延长呈递增趋势.(5)相关性分析显示:RhoA与P27mRNA的表达无明显相关(r=-0.105);RhoA与P27蛋白的表达呈显著负相关(r=-0.943,P<0.01).结论:MSCs抑制HSCs增殖的机制可能是通过RhoA-P27通路调控HSCs细胞周期改变;RhoA活性的下调可能是引起HSCs内P27蛋白表达增加的原因.AIM： To investigate the effects of bone marrow mesenchymal stem cells （MSCs） on the mRNA and protein expression of RhoA （Ras homolog gene family member A） and P27 in hepatic stellate cells （HSCs） and explore the mechanisms how MSCs regulate cell cycle progression of HSCs.METHODS： MSCs were isolated from rat bone marrow and propagated in culture flasks. Meanwhile, HSCs and fibroblasts were thawed and passaged. An indirect co-culture system between MSCs/fibroblasts and HSCs was established using a Transwell membrane system （diameter：24 mm; pore size： 0.4 μm）. HSCs were randomly divided into three groups： blank control group （HSCs alone）, negative control group （HSCs plus fi broblasts）, and experimental group （HSCs plus MSCs）. Cell proliferation was tested by WST-8 assay. Cell-cycle phases were determined by flow cytometry. The mRNA and protein expression of RhoA and P27 in HSCs was determined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively. RESULTS： After 24 hours of co-culture, the reduced rate of cell proliferation in the experimental group was signif icantly higher than those in the blank control group and negative control group co-culture（both P 〈 0.01）. Flow cytometry analysis showed that, after 12 hours of co-culture, the percentage of HSCs in the G0/G1 phase in the experimental group was significantly higher than those in the two control groups （both P 〈 0.01）, while the percentage of HSCs in the S phase in the experimental group was signif icantly lower than those in the two control groups （both P 〈 0.01）. After 12 hours of co-culture, the expression level of RhoA mRNA in the experimental group was signif icantly lower than those in the two control groups （both P 〈 0.01）, whereas the expression level of P27 mRNA showed no signif icant differences between the experimental group and the two control groups （both P 〉 0.05）. The expression level of RhoA protein in the experimental group was signi

关 键 词：骨髓间充质千细胞 肝星状细胞 RHOA P27 细胞周期 

分 类 号：R575.2[医药卫生—消化系统] R329.2[医药卫生—内科学]