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目的 探讨以重组慢病毒为载体转染Klotho基因（抗衰老基因）对SD大鼠心肌中骨髓间充质干细胞（bone marrow Mesenchymal stem cells,BMSCs）存活率及其可能的机制。方法 全骨髓贴壁培养法体外培养SD大鼠BMSCs；以重组慢病毒为载体将KLotho基因转染至大鼠BMSCs，用RT-PCR鉴定BMSCs中Klotho基因mRNA的表达；腹腔连续注射异丙肾上腺素建立SD大鼠心衰模型；并随机分为3组，即EGFP-Klotho-BMSCs组（慢性心衰SD大鼠尾静脉注射Klotho基因修饰的BMSCs），EGFP-BMSCs组（慢性心衰SD大鼠尾静脉注射空载慢病毒转染的BMSCs），生理盐水组（慢性心衰SD大鼠尾静脉注射与等容积的生理盐水）。细胞移植治疗4周后，在荧光显微镜下观察各组大鼠心肌组织绿色荧光蛋白的表达情况，RT-PCR检测各组大鼠心肌组织中Klotho基因的表达。结果 ①全骨髓贴壁法成功培养及传代BMSCs；②以重组慢病毒为载体成功将Klotho基因转染到BMSCs，荧光显微镜下第4天的细胞转染效率达70%~80%以上；③RT-PCR结果表明与EGFP-BMSCs组比较，EGFP-Klotho-BMSCs组的BMSCs中Klotho基因表达明显增多（P<0.05）；④干细胞移植4周后，EGFP-Klotho-BMSCs组和EGFP-BMSCs组大鼠心肌组织中仍可见EGFP绿色荧光表达，且EGFP-Klotho-BMSCs组心肌组织中绿色荧光表达数量较EGFP-BMSCs组显著增多。结论 转染Klotho基因可有效提高BMSCs在SD大鼠心肌中的存活率，其可能的机制为Klotho基因具有抗细胞凋亡及改变心肌内环境的作用。
Objective To investigate the survival rate of bone marrow mesenchymal stem cells (BMSCs) transfected with Klotho gene (anti-aging gene) using recombinant lentivirus as a vector and its possible mechanism. Methods BMSCs were cultured in vitro by whole bone marrow adherent culture. KLotho gene was transfected into BMSCs using recombinant lentivirus as a vector. The expression of Klotho mRNA in BMSCs was identified by RT-PCR. Intraperitoneal injection of isoproterenol Sprague-Dawley rats were randomly divided into three groups: EGFP-Klotho-BMSCs group (Klotho gene-modified BMSCs injected into tail vein of chronic heart failure SD rats), EGFP-BMSCs group Tail vein injection of empty lentiviral transfection of BMSCs), saline group (chronic ventricular fibrillation SD rats tail vein injection and equal volume of saline). Four weeks after the cell transplantation, the expression of green fluorescent protein (GFP) in the myocardium of each group was observed under a fluorescence microscope. The Klotho gene expression in each group was detected by RT-PCR. Results ①The BMSCs were successfully cultured and passaged by the whole bone marrow adherent method. ② The Klotho gene was successfully transfected into BMSCs with the recombinant lentivirus as a vector. The transfection efficiency was over 70% ~ 80% PCR results showed that compared with EGFP-BMSCs group, the expression of Klotho gene in BMSCs of EGFP-Klotho-BMSCs group was significantly increased (P <0.05); ④ After 4 weeks of stem cell transplantation, EGFP-Klotho-BMSCs group and EGFP-BMSCs group EGFP green fluorescence was still observed in myocardial tissue, and the number of green fluorescent protein in myocardial tissue of EGFP-Klotho-BMSCs group was significantly higher than that of EGFP-BMSCs group. Conclusion Transfection of Klotho gene can effectively improve the survival rate of BMSCs in the SD rat myocardium. The possible mechanism is that Klotho gene has anti-apoptosis effect and changes the myocardial environment.