PTEN基因的沉默对骨髓间充质干细胞体外生物学特性和体内移植治疗心肌梗死的影响

Effect of silencing of PTEN gene on Mesenchymal Stem Cells biology property in vitro and transplantation to treat myocardial infarction in vivo

目的:研究PTEN基因沉默对骨髓间充质干细胞生物学性状的影响,并观察其在心肌梗死区域的存活和增殖情况。为干细胞移植治疗心肌梗死提供更优良的种子细胞。方法:通过脂质体法将PTEN基因特异的siRNA转染入骨髓间充质干细胞。RT-PCR法和Western blot法检测PTEN基因RNAi前后的mRNA和蛋白表达的变化。体外实验采用MTT法描绘细胞生长曲线,流式细胞仪(PI单染法)检测细胞周期,制备细胞缺血缺氧模型,流式细胞仪(Annexin V和PI双染法)检测各组细胞对缺血缺氧的耐受情况。体内实验通过制备动物心肌梗死模型,移植骨髓间充质干细胞,采用荧光显微镜观察细胞的存活和增殖情况。结果:RNAI组的PTEN基因的mRNA和蛋白表达均明显下降,PTEN基因被明显抑制。体外实验:MTT法显示RNAI组的细胞生长活力明显增强。RNAI组的细胞周期向右偏移。RNAI组对缺血缺氧的耐受能力明显增强。体内实验:荧光显微镜显示RNAI组移植的骨髓间充质干细胞在心肌梗死区域的数量明显增多。结论:PTEN基因的沉默能有效地促进骨髓间充质干细胞的增殖,使细胞周期向右偏移,同时能抑制细胞凋亡,增强细胞对缺血缺氧的耐受能力。体内实验也证实,PTEN基因的沉默后的骨髓间充质干细胞更容易在心肌梗死区域存活和增殖,为干细胞移植治疗心肌梗死提供新的思路。

Objective：To investigate the biological effect of provoked gene silencing of PTEN on the Mesenchymal Stem Cells,and to observe survival and proliferation of cells in the Injured Myocardium,and may find the better seeds cells for Transplanting to treat myocardial infarction.Method：We transfect specific siRNA of PTEN gene into the Mesenchymal Stem Cells by the liposome.Detect the change of the mRNA and protein of PTEN by the way of RT-PCR and Western blot in the anterior-posterior transfection.in vitro,we trace the cell growth curve by MTT,and detect Cell Cycle using flow cytometry（PI simple staining）,also,we detect cells tolerance to ischemia and anoxemic using flow cytometry（Annexin V and PI double staining） in the cell model of ischemia and anoxemic.in vivo,we prepare the animal model of myocardial infarction,then transplant the Mesenchymal Stem Cells into the Injured Myocardium by local injection,eventually,we observe the cell survival and proliferation by fluorescence microscope.Result：RT-PCR and Western blot showed that the mRNA and protein expression of PTEN significantly decreased in the group of RNAi.MTT assay indicated that RNAi of PTEN gene dramatically promoted cellular proliferation,and obviously strengthened cellular tolerance of ischemia and hypoxia.In vivo,the fluorescence microscope showed that the survival rate of the Mesenchymal Stem Cell in myocardial infarction region increase obviously in the group of RNAi.Conclusion：PTEN gene silencing by RNAi can promote effectively proliferation of the Mesenchymal Stem Cell with the cell cycle curve shifting right,and reinforce the tolerance of ischemia and anoxemic by inhibit cell apoptosis.In vivo,the Mesenchymal Stem Cell after PTEN gene silencing can preferably survive and proliferate in the region of myocardial infarction,and may provide a new therapeutic strategy for myocardial infarction by stem cell transplantation.