| 慢病毒载体过表达Klotho基因对骨髓间充质干细胞在慢性心衰大鼠心肌中存活率的影响 |
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| 引用本文： | 朱向情,杨晓华,潘兴华. 慢病毒载体过表达Klotho基因对骨髓间充质干细胞在慢性心衰大鼠心肌中存活率的影响[J]. 心脏杂志, 2017, 29(6): 641-646 |
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| 作者姓名： | 朱向情 杨晓华 潘兴华 |
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| 作者单位： | （1.昆明总医院地方干部病房，云南 昆明 650032; |
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| 摘 要： | 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。
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| 关 键 词： | 心力衰竭 Klotho基因 骨髓间充质干细胞 |
| 收稿时间： | 2016-09-26 |
Effect of mesenchymal stem cells transfected with lentiviral vector over-expressing Klotho on stem cell survival in a rat myocardium heart failure model |
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| Abstract: | AIM To observe whether bone-derived mesenchymal stem cells(BMSCs)infected with lentiviral vectors bearing Klotho gene will increase the stem cell survival in a rat myocardium of heart failure.METHODS Rat BMSCs were isolated,cultured,and purified by whole bone marrow adherence method for morphology observations. Lentiviral vectors bearing EGFP-Klotho were constructed. The expression of Klotho mRNA in the BMSCs was detected by RT-PCR. SPF SD rats with heart failure (HF) created by subcutaneous injections of isoproterenol (ISO) were randomly divided into three groups: saline-control HF group, BMSCs with transfected Klotho gene group, and BMSCs with transfected EGFP group. Green fluorescin expression was observed on frozen heart section and expression of Klotho mRNA in the rat myocardium of heart failure was detected by RT-PCR. RESULTS BMSCs were successfully cultured by whole bone marrow adherent method and lentiviral vectors over-expressing Klotho were constructed successfully. BMSCs were infected with lentivirus bearing Klotho and the transfection efficiency was above 70%-80% on day 4 after transfection. Green fluorescin distributed around in myocardial tissues was seen in Klotho-BMSCs group and EGFP-BMSCs group 4 weeks after transfection.RT-PCR detection results showed that Klotho mRNA was expressed in the rat myocardium in each group. The expression level in EGFP-Klotho-BMSCs group was significantly higher than that in EGFP-BMSCs group (P<0.05). CONCLUSION Klotho transfection can increase the survival of BMSCs in rat myocardium of heart failure, possibly by its resistance to apoptosis and improvement of myocardial en |
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