小分子干扰RNA介导RhoA沉默优化骨髓间充质干细胞的培养  

Optimization of small interfering RNA-mediated RhoA silencing in bone marrow mesenchymal stem cell cultures

作　　者：吕刚[1] 姚鑫[2] 

机构地区：[1]天津医科大学研究生院,天津市300000 [2]天津市环湖医院神经外科,天津市300000

出　　处：《中国组织工程研究与临床康复》2010年第45期8365-8368,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:以往骨髓间充质干细胞的培养方法存在衰老和分化率低等问题。目的:检测是否可以通过沉默RhoA基因的方法优化骨髓间充质干细胞培养。方法:体外培养SD大鼠骨髓间充质干细胞,经小分子干扰RNA转染以沉默RhoA基因表达,分为3组培养:干细胞组(未转染小分子干扰RNA)、经随机打乱顺序的小分子干扰RNA转染干细胞组、经小分子干扰RNA转染的干细胞组。用RT-PCR,Westernblot检测骨髓间充质干细胞在转染前后RhoA基因和蛋白的表达。应用细胞生长曲线、MTT比色法观察细胞生长的优化作用,采用流式细胞术测定细胞周期分布的变化。结果与结论:与干细胞组、经随机打乱顺序的小分子干扰RNA转染干细胞组比较,经小分子干扰RNA转染的干细胞组RhoA基因和蛋白表达量明显降低(P<0.05),细胞的生长速度明显增快,细胞周期G0/G1期减少,S期细胞数增多(P<0.05)。说明通过沉默RhoA基因的方法可以促进骨髓间充质干细胞增殖,优化培养方法。BACKGROUND： Previous culture method of bone marrow mesenchymal stem cells （BMSCs） has problems such as senescence and low differentiation rate. OBJECTIVE： To determine whether the RhoA gene silencing can optimize BMSC cultures. METHODS： BMSCs from Sprague Dawley rats were cultured in vitro, and were transfected by small interfering RNA （siRNA） to knock down the expression of RhoA. BMSCs were divided into three groups： stem cell group （non-transfection of siRNA）, disrupted by random sequence siRNA transfection stem cell group, and siRNA transfection of stem cell group. Reverse transcription-pelymerase chain reaction （RT-PCR） and Western blot assay were used to assess RhoA gene and protein expression before and afler transfection, Cellular proliferation optimization was determined by the cell growth curve and MTT assay. The cell cycle was analyzed by flow cytometry. RESULTS AND CONCLUSION： Expression of RhoA gene and protein was significantly reduced following transfection of siRNA compared with stem cell group （P 〈 0.05）. The cell growth speed was significantly increased, the cell cycle of Go/G1 phase was decreased and cell number in S phase was increased （P 〈 0.05）. These indicated that the RhoA gene silencing can promote BMSC proliferation, and optimize culture methods.

关 键 词：细胞培养 骨髓间充质干细胞 RHOA RNA干扰 干细胞培养 

分 类 号：R394.2[医药卫生—医学遗传学]