摘要
背景:肾上腺髓质素基因转染能增强骨髓间充质干细胞在缺血、缺氧环境下的抗凋亡能力,但其机制尚未完全明确。目的:观察肾上腺髓质素对骨髓间充质干细胞缺血、缺氧时Bax、Bcl-2和Caspase-3等凋亡相关调节蛋白表达的影响。方法:贴壁法分离、培养、纯化SD大鼠骨髓间充质干细胞,缺氧、无血清培养0,3,6,9,12 h后用Western blot测定Bax、Bcl-2、Caspase-3蛋白的表达以确定最佳缺氧时间。采用缺氧6 h为时间点,根据缺氧、无血清培养前是否进行肾上腺髓质素预处理分为对照组(未加入肾上腺髓质素)及肾上腺髓质素不同质量浓度组(1,10,100μg/L),然后采用Western blot测定Bax、Bcl-2、Caspase-3蛋白的表达。结果与结论:1无血清培养条件下缺氧0,3,6,9,12 h后骨髓间充质干细胞Bax、Bcl-2表达均增加(P<0.05),缺氧6 h时Bax/Bcl-2比值、caspase-3蛋白达到最小(P<0.05)。2缺氧6 h时,与1,10μg/L肾上腺髓质素组相比,100μg/L肾上腺髓质素组表达Bax蛋白、Caspase-3蛋白最低,Bax/Bcl-2比值最低,而表达Bcl-2蛋白最高(P<0.05)。结果表明肾上腺髓质素能降低无血清、缺氧条件下骨髓间充质干细胞Bax/Bcl-2比值、Caspase-3蛋白表达,该效应存在剂量依赖性。
BACKGROUND：Adrenomedulin gene transfection can strength the anti-apoptotic ability of bone marrow mesenchymal stem cels under ischemia and hypoxia, but its mechanism is not yet clear. OBJECTIVE：To investigate the effect of adrenomedulin on the expression of apoptosis-related proteins, Bax, Bcl-2 and Caspase-3, in bone marrow mesenchymal stem cels under hypoxia and ischemia. METHODS：Bone marrow mesenchymal stem cels of Sprague-Dawley rats were isolated, cultured and purified, and then cultured in serum-free medium under hypoxic condition for 0, 3, 6, 9, 12 hours. Then, western blot assay was employed to detect the expression of Bax, Bcl-2 and Caspase-3 so as to determine the optimal hypoxia time that was determined at 6 hours of hypoxia. Depending on whether adrenomedulin pretreatment was done, the cels were divided into control group （with no adrenomedulin pretreatment before hypoxia and ischemia） and adrenomedulin groups with different concentrations （1, 10, 100 μg/L）. Afterwards, the expression of Bax, Bcl-2 and Caspase-3 was detected by using western blot assay. RESULTS AND CONCLUSION：（1） After cultured in serum-free medium under hypoxia for 0, 3, 6, 9, 12 hours, the expression of Bax, Bcl-2 and Caspase-3 in bone marrow mesenchymal stem cels were increased （P 〈 0.05）;at 6 hours of hypoxia, the Bax/Bcl-2 ratio and Caspase-3 expression reached the minimum value （P 〈 0.05）. （2） At 6 hours of hypoxia, the expression of Bax and Caspase-3 protein as wel as Bax/Bcl-2 ratio became the lowest in the 100 μg/L group compared with the 1 and 10 μg/L groups, but the expression of Bcl-2 protein reached the peak （P 〈 0.05）. These findings indicate that adrenomedulin can reduce the expression of Bax/Bcl-2 ratio and Caspase-3 protein in bone marrow mesenchymal stem cels cultured in serum-free medium under hypoxic conditions, which is in a dose-dependent manner.
出处
《中国组织工程研究》
CAS
北大核心
2015年第32期5092-5096,共5页
Chinese Journal of Tissue Engineering Research
基金
福建省卫生厅青年基金(2011-1-15
2011-1-32)
福建省自然科学基金(2014J01433)~~
