肾上腺髓质素基因转染改善缺氧培养骨髓间充质干细胞抗凋亡能力

发布时间：2019-02-21 09:39

【摘要】： [目的]研究肾上腺髓质素(ADM)基因转染对骨髓间充干细胞(MSCs)在缺氧及无血清培养条件下的增殖、凋亡、分泌血管内皮生长因子(VEGF)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。骨髓间充质干细胞分为未转染组,空载体组,Ad-ADM转染组。在缺氧、无血清条件下进行骨髓间充质干细胞的培养,在缺氧0、3、6、9、12、16、20、24h CCK-8检测细胞增殖、ELISA检测检测细胞上清液肾上腺髓质素及血管内皮生长因子的含量、Tunel法检测细胞凋亡。 [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%。缺氧、无血清培养条件下,3组骨髓间充质干细胞均出现生长抑制、凋亡增加,但是肾上腺髓质素基因转染组于0,3,6,9、12、16h细胞凋亡率显著低于其它2组(P0.05)。缺氧9,12h,骨髓间充质干细胞分泌肾上腺髓质素及血管内皮生长因子达到高峰,且Ad-ADM转染组显著增高于其它2组(P0.05)。 [结论]在缺氧、无血清培养条件下(20h),肾上腺髓质素基因转染增强骨髓间充质干细胞抗凋亡能力,这可能与肾上腺髓质素、血管内皮生长因子表达增加有关。
[Abstract]:[objective] to study the effect of adrenomedullin (ADM) gene transfection on proliferation, apoptosis and secretion of vascular endothelial growth factor (VEGF) in bone marrow mesenchymal stem cells (MSCs) in hypoxic and serum-free culture. [methods] Rat bone marrow mesenchymal stem cells were isolated and cultured by adherent method in vitro. The infection rate of recombinant adenovirus containing adrenomedullin gene (Ad-ADM) on bone marrow mesenchymal stem cells (BMSCs) was detected by x-gal staining. Bone marrow mesenchymal stem cells were divided into untransfected group, empty vector group and Ad-ADM transfection group. Bone marrow mesenchymal stem cells (BMSCs) were cultured in anoxic and serum-free condition. Cell proliferation was detected by CCK-8 at 24 h after hypoxia, and the content of adrenomedullin and vascular endothelial growth factor (VEGF) in supernatant was detected by ELISA. Apoptosis was detected by Tunel. [results] the transfection efficiency of recombinant adenovirus on bone marrow mesenchymal stem cells was dose-dependent with the infection of complex (MOI), and the infection rate reached 95.4when MOI was 150. Under the condition of anoxia and serum-free culture, the growth inhibition and apoptosis of bone marrow mesenchymal stem cells in all three groups were inhibited, but the apoptotic rate in the adrenomedullin gene transfection group was significantly lower than that in the other two groups (P0.05). The secretion of adrenomedullin and vascular endothelial growth factor (VEGF) from bone marrow mesenchymal stem cells reached the peak at 9h after hypoxia, and the levels of adrenomedullin and vascular endothelial growth factor (VEGF) in the Ad-ADM transfected group were significantly higher than those in the other two groups (P0.05). [conclusion] under hypoxic and serum-free culture conditions (20 h), adrenomedullin gene transfection enhances the anti-apoptosis ability of bone marrow mesenchymal stem cells, which may be related to the increased expression of adrenomedullin and vascular endothelial growth factor.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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