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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 effect of PTEN gene silencing on the biological characteristics of bone marrow mesenchymal stem cells and to observe its survival and proliferation in myocardial infarction area. Stem cell transplantation for the treatment of myocardial infarction provide better seed cells. Methods: PTEN gene-specific siRNA was transfected into bone marrow mesenchymal stem cells by liposome method. The mRNA and protein expressions of PTEN gene before and after RNAi were detected by RT-PCR and Western blot. MTT assay was used to characterize the cell growth curve, flow cytometry (PI single staining) was used to detect the cell cycle, cell hypoxia-ischemia model was established, and the cell pairs were detected by flow cytometry (Annexin V and PI double staining) Ischemia and hypoxia tolerance. In vivo experiments by animal model of myocardial infarction, transplantation of bone marrow mesenchymal stem cells, the use of fluorescence microscopy of cell survival and proliferation. Results: The mRNA and protein expressions of PTEN gene in RNAi group were significantly decreased, PTEN gene was significantly inhibited. In vitro experiments: MTT assay showed that RNAi group cell viability significantly increased. The cell cycle of the RNAI group shifted to the right. RNAi group of hypoxia tolerance increased significantly. In vivo experiments: Fluorescent microscopy showed a significant increase in the number of MSCs transplanted in the RNAI group in the infarcted area. Conclusion: Silencing PTEN can effectively promote the proliferation of BMSCs, shift the cell cycle to the right, inhibit apoptosis and enhance the tolerance of cells to hypoxia and hypoxia. In vivo experiments also confirmed that PTEN gene silencing of bone marrow mesenchymal stem cells more likely to survive and proliferate in myocardial infarction area, providing a new idea for stem cell transplantation in the treatment of myocardial infarction.