肾上腺髓质素基因转染改善缺氧培养骨髓间充质干细胞的抗凋亡能力

Adrenomedullin gene transfection improves anti-apoptotic ability of bone marrow mesenchymal stem cells under hypoxic conditions

背景:基因转染可以提高骨髓间充质干细胞在移植区的存活,但目前对肾上腺髓质素基因转染骨髓间充质干细胞在缺氧条件下增殖、凋亡及分泌情况的研究很少。目的:观察肾上腺髓质素基因转染对骨髓间充质干细胞在缺氧及无血清培养条件下增殖、凋亡、分泌血管内皮生长因子的影响。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的感染率。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0,3,6,9,12,16,20,24hCCK-8法检测细胞增殖情况,ELISA法检测检测细胞上清液肾上腺髓质素及血管内皮生长因子表达,TUNEL法检测细胞凋亡情况。结果与结论: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具有量效关系,病毒感染复数为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他两组(P﹤0.05)。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他两组(P﹤0.05)。提示在缺氧、无血清培养条件下(<20h),肾上腺髓质素基因转染可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。

BACKGROUND：Gene transfection can elevate the survival of bone marrow mesenchymal stem cells （BMSCs） in transplanted region,but there are a few reports addressing proliferation,apoptosis and secretion of adrenomedullin （ADM） gene-transfected BMSCs under hypoxic condition. OBJECTIVE：To observe effects of ADM gene transfection on BMSCs proliferation,apoptosis and secretion of vascular endothelial growth factor under hypoxic and serum-free culture conditions. METHODS：Rat BMSCs were isolated and amplified in vitro by differential adhesion method. The infection efficiency of recombinant adenovirus Ad-ADM in BMSCs was tested by x-gal staining. BMSCs were divided into non-transfection group,empty vector group and Ad-ADM transfection group. BMSCs cultured under hypoxia and serum-free conditions for 0,3,6,9,12,16,20,24 hours respectively. Cell proliferation was detected by cell counting kit-8 assay. ADM and vascular endothelial growth factor in culture medium were detected by enzyme-linked immunosorbent assay. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling. RESULTS AND CONCLUSION：Infection rate of BMSCs increased with multiplicity of infection （MOI） in a dose-dependant manner. When MOI was 150,the infection rate was 95.4%. After BMSCs cultured in hypoxia and serum-free medium,cell growth inhibition and apoptosis increased,but the apoptotic rate in Ad-ADM transfection group was significantly lower than other 2 groups after cultured for 0,3,6,9,12,16 hours （P 0.05）. At 9 and 12 hours,BMSCs secreted ADM and vascular endothelial growth factor reached a peak and were significantly higher in the Ad-ADM transfection group than other two groups （P 0.05）. Results have indicated that under hypoxia and serum-free culture conditions （ 20 hours）,ADM gene transfection enhances the anti-apoptotic ability of BMSCs,which may be associated with the increased expression of ADM and vascular endothelial growth factor.