肾上腺髓质素基因转染的骨髓间充质干细胞缺氧共培养对新生大鼠心肌细胞凋亡的影响

发布时间：2018-01-08 05:03

本文关键词：肾上腺髓质素基因转染的骨髓间充质干细胞缺氧共培养对新生大鼠心肌细胞凋亡的影响　出处：《福建医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：[目的]研究肾上腺髓质素（ADM）基因转染的骨髓间充质干细胞（MSCs）缺氧共培养对新生大鼠心肌细胞凋亡的影响。 [方法]贴壁法体外分离、扩增培养大鼠骨髓间充质干细胞。用x-gal染色测含肾上腺髓质素基因的重组腺病毒(Ad-ADM)对骨髓间充质干细胞感染率。乳鼠心肌细胞分离、纯化、培养与鉴定。乳鼠心肌细胞缺氧、无血清条件下培养3、6、12、24小时并检测其凋亡率。用Transwell小室缺氧、无血清条件下共培养骨髓间充质干细胞和乳鼠心肌细胞，24小时后检测共培养体系上清液中肾上腺髓质素含量并检测乳鼠心肌细胞凋亡率。实验分组为：Ad-ADM转染共培养组，Ad-Null转染共培养组，wildtype共培养组，同时设置单独乳鼠心肌细胞缺氧、无血清培养组。 [结果]重组腺病毒对骨髓间充质干细胞的转染率与病毒感染复数（MOI）具有量效关系，MOI为150时细胞的感染率达95.4%。在缺氧、无血清培养条件下，乳鼠心肌细胞在缺氧3h、6h、12h、24h的凋亡率逐步升高，各时间点凋亡率差异具有统计学意义（P＜0.001）。三组共培养体系中乳鼠心肌细胞凋亡率明显低于单独乳鼠心肌细胞缺氧培养组（P＜0.05）；Ad-ADM转染共培养组乳鼠心肌细胞凋亡率低于空载体转染、共培养组和未转染、共培养组（P＜0.05）。空载体转染、共培养组和未转染、共培养组相比，，乳鼠心肌细胞凋亡差异未达到统计学意义。肾上腺髓质素基因转染共培养组的上清液中肾上腺髓质素分泌量高于空载体转染共培养组和未转染共培养组（P＜0.05）。空载体转染共培养组与未转染共培养组相比，肾上腺髓质素差异未达到统计学意义。 [结论]与骨髓间充质干细胞共培养可以降低缺氧、无血清培养条件下乳鼠心肌细胞的凋亡率。与未转染的骨髓间充质干细胞相比，在缺氧、无血清培养条件下（24小时），肾上腺髓质素基因转染可以增强共培养对乳鼠心肌细胞的凋亡的影响，这可能与肾上腺髓质素表达增加有关。
[Abstract]:[Objective] to study the effect of hypoxia co-culture of bone marrow mesenchymal stem cells (MSCs) transfected with adrenal medullary hormone (ADM) gene on cardiomyocyte apoptosis in neonatal rats. [Methods] the adherent method was isolated in vitro. Bone marrow mesenchymal stem cells (BMSCs) were amplified and cultured in rats. The infection rate of the recombinant adenovirus Ad-ADM containing adrenomedullin gene was detected by x-gal staining. Purification, culture and identification. Neonatal rat cardiomyocytes were exposed to hypoxia and cultured for 24 hours without serum. The apoptosis rate was measured by Transwell chamber hypoxia. Bone marrow mesenchymal stem cells and neonatal cardiomyocytes were co-cultured in serum-free medium. After 24 hours, the content of adrenomedullin in supernatant of co-culture system and the rate of cardiomyocyte apoptosis in neonatal rats were detected. Wildtype co-culture group, at the same time set up isolated neonatal rat cardiomyocytes hypoxia, serum-free culture group. [Results: the transfection efficiency of recombinant adenovirus on bone marrow mesenchymal stem cells was dose-dependent and the infection rate was 95.4 when moi was 150. Under the condition of serum-free culture, the apoptosis rate of neonatal rat cardiomyocytes increased gradually after hypoxia for 3 h, 6 h and 12 h for 24 h. There was significant difference in apoptosis rate at different time points (P < 0.001). The apoptosis rate of neonatal rat cardiomyocytes in the co-culture system was significantly lower than that in hypoxic culture group (P < 0.05). The apoptosis rate of neonatal rat cardiomyocytes in Ad-ADM transfection group was lower than that in empty vector transfection group, and the co-culture group and non-transfection group (P < 0.05), empty vector transfection, co-culture group and non-transfection group. Compared with co-culture group. The amount of adrenomedullin secreted in the supernatant of the co-culture group was higher than that in the empty vector co-culture group and the untransfected co-culture group. P < 0.05. The empty vector transfection co-culture group was compared with the non-transfection co-culture group. The difference of adrenomedullin was not statistically significant. [Conclusion: Co-culture with bone marrow mesenchymal stem cells can reduce the apoptosis rate of neonatal rat cardiomyocytes in anoxic and serum-free culture, compared with untransfected bone marrow mesenchymal stem cells. In serum-free culture for 24 hours, adrenomedullin gene transfection could enhance the effect of co-culture on cardiomyocyte apoptosis in neonatal rats, which may be related to the increase of adrenomedullin expression.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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