Cardioprotection by hypoxia-induced rat adipose-derived stem cells through paracrine mechanisms

doi:10.3969/j.issn.2095-4344.0752

Abstract

Abstract:

BACKGROUND: Adipose-derived stem cells (ADSCs) represent one of the promising cell sources for myocardial regeneration due to their easy accessibility and efficacy in the improvement of cardiac function following myocardial infarction. However, previously reported studies on the underlying mechanism of ADSCs-mediated cardioprotective effect mainly focused on the ADSCs cultured at room air.
OBJECTIVE: To test the paracrine actions and anti-apoptotic effect of ADSCs under hypoxic conditions.
METHODS: After isolation and culture, neonatal rat myocardial cells were injured by hydrogen peroxide and co-cultured with rat ADSCs under normoxia and hypoxia (10% O2) conditions. Ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in the cell pellet and levels of vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and basic fibroblast growth factor (bFGF) were tested by ELISA. Expression of apoptotic proteins Bax and Bcl-2 were determined by western blot.
RESULTS AND CONCLUSION: GSH/GSSG, VEGF, IGF-1, and bFGF were decreased in neonatal rat myocardial cells injured by hydrogen peroxide. ADSCs significantly attenuated hydrogen peroxide-induced myocardial apoptosis by increasing the ratio of GSH/GSSG and the secretion of VEGF, IGF-1 and bFGF. ADSCs also down-regulated Bax expression and up-regulated Bcl-2 expression. To conclude, hypoxic conditions can enhance the anti-apoptosis and cardioprotective effects of ADSCs through the paracrine mechanism.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Adipose Tissue, Myocytes, Cardiac, Coculture Techniques, Cell Hypoxia, Tissue Engineering

CLC Number:

R394.2

Gao Yu-ping, Lin Yuan-yuan, Li Xue-wen, Fan Chun-hui, Yang Fan, Hao Da-jie, Ge Wen-jia. Cardioprotection by hypoxia-induced rat adipose-derived stem cells through paracrine mechanisms[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1344-1349.

Figures/Tables 2

2.1 脂肪干细胞及心肌细胞的形态图1是不同分组处理下脂肪干细胞及心肌细胞的形态，传代后的脂肪干细胞形态比较均一，呈长梭形，漩涡样生长，大鼠乳鼠心肌细胞伪足相互接触交织成密集的网，形成放射状排列的细胞簇，经过100 μmol/L过氧化氢处理后心肌细胞死亡明显，而在低氧状态下与脂肪干细胞共培养后，心肌细胞死亡较前有所减少。 2.2 细胞沉淀中还原型谷胱甘肽、氧化型谷胱甘肽水平由图2A可见过氧化氢损伤心肌细胞后，心肌细胞中还原型谷胱甘肽/氧化型谷胱甘肽比值明显下降(P < 0.05)，正常氧体积分数时，脂肪干细胞与心肌细胞共培养不能产生抗氧化作用(P > 0.05)；而在低氧状态下，损伤心肌细胞与脂肪干细胞共培养可以使还原型谷胱甘肽/氧化型谷胱甘肽比值增加(P < 0.05，图2B)。 2.3 培养基上清中细胞因子水平 2.3.1 培养基上清中血管内皮生长因子水平图3A中乳鼠心肌细胞与脂肪干细胞共培养后血管内皮生长因子水平明显上升，同时，损伤的乳鼠心肌细胞与脂肪干细胞共培养后培养基上清中血管内皮生长因子水平也明显上升(P < 0.001)，说明脂肪干细胞对损伤的心肌细胞有一定的保护作用。由图3B可以看出，低氧状态下共培养细胞的培养基上清中血管内皮生长因子水平明显高于损伤心肌细胞(P < 0.001)。 2.3.2 培养基上清中胰岛素样生长因子水平图4A可见，过氧化氢处理心肌细胞后胰岛素样生长因子水平降低，损伤心肌细胞与脂肪干细胞共培养后，胰岛素样生长因子水平上升，但统计结果显示差异无显著性意义。图4B可见，在低氧状态下，与损伤的乳鼠心肌细胞组相比，共培养组培养基上清中胰岛素样生长因子水平明显上升(P < 0.01)，说明脂肪干细胞在低氧状态下可以促进胰岛素样生长因子的分泌。 2.3.3 培养基上清中碱性成纤维细胞生长因子水平图5A可见正常乳鼠心肌细胞与脂肪干细胞共培养后碱性成纤维细胞生长因子水平明显上升，在过氧化氢处理心肌细胞后，几乎没有碱性成纤维细胞生长因子的表达。当损伤心肌细胞与脂肪干细胞共培养后，碱性成纤维细胞生长因子水平明显上升(P < 0.05)，提示脂肪干细胞促进碱性成纤维细胞生长因子的分泌。图5B可见低氧状态下共培养组培养基中碱性成纤维细胞生长因子水平明显增加(P < 0.001)。 2.4 细胞沉淀中Bax和Bcl-2蛋白表达图6为不同氧体积分数下脂肪干细胞与损伤的乳鼠心肌细胞共培养时Bax和Bcl-2蛋白的表达。正常氧体积分数下，过氧化氢损伤的心肌细胞与脂肪干细胞共培养后，Bax蛋白表达下降(P < 0.05)，Bcl-2蛋白表达上升(P < 0.001)。与正常氧体积分数下培养相比，在低氧状态下，损伤心肌细胞与脂肪干细胞共培养之后，Bax的表达有所下降，Bcl-2的表达明显上升，可以看到在共培养体系中，脂肪干细胞对心肌细胞的凋亡有保护作用，而低氧状态能够增强脂肪干细胞抗凋亡作用。"

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