Akt基因转染对骨髓间充质干细胞缺氧时凋亡和增殖的影响

目的采用Akt基因转染鼠骨髓MSCs探讨Akt基因是否减轻MSCs缺氧时的凋亡和提高缺氧时的增殖能力,即耐缺氧能力。方法将转染和未转染Akt基因的MSCs置于94%N2、1%O2和5%CO2缺氧箱中37℃孵育不同时间（常氧、缺氧0.5h、1h、2h、4h和8h）后,Annexin V/PI双染法行流式细胞仪分析凋亡率（apoptoticrate,AR）和死亡率（deadrate,DR）、MTT法分析细胞增殖状态、Rt-PCR和Western blot等检测Akt和p-Akt表达以及放射同位素法检测MSCs对氚标-葡萄糖（^3H-G）的摄取等。结果1.Akt基因显著降低MSCs缺氧时AR和DR（P〈0.01）,而各缺氧时间点没有统计学意义（P〉0.05）;2.Akt基因显著增高MSCs常氧和缺氧（与未转染Akt基因MSCs同等条件下比较）时增殖能力（P〈0.01）,缺氧时增殖能力显著低于常氧时（P〈0.01）;3.Akt基因显著增高常氧时MSCsAkt mRNA（P〈0.01）和蛋白（P〈0.01）表达,而不增高p-Akt蛋白（P〉0.05）表达;Akt基因显著提高缺氧时p-Akt蛋白（P〈0.01）表达,而不提高常氧时p-Akt蛋白（P〉0.05）表达;4.Akt基因显著增高MSCs常氧和缺氧（与未转染Akt基因MSCs同等条件下比较）时^3H-G的摄取（P〈0.01）,缺氧时^3H-G的摄取显著性低于常氧培养时（P〈0.01）;^3H-G的摄取与细胞增殖显著正相关（r=0.79,P=0.015）而与细胞凋亡显著负相关（r=-1.47,P=0.023）。结论Akt基因转染可显著提高MSCs耐缺氧能力,此可能与缺氧时改善MSCs葡萄糖摄取等有关。

APOPTOSIS AND PROLIFERATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS TRANSFECTED BY AKT GENE UNDER HYPOXIA

Objective To elucidate the effects of Akt gene transfection on apoptosis, proliferation and glucose uptake of bone marrow mesenchymal stem cells （MSCs） of Wistar rats, under hypoxia. Materials and Methods MSCs with or withont Akt gene transfection were cultured in chambers with 94%N2, 1%O2, 5%CO2 at 37℃ for different durations （normoxia, and hypoxia of 0. 5 h, 1 h, 2 h, 4 h-8 h）. Then the apoptotic rate （AR） and death rate （DR） were analyzed by flow cytometry after Annexin V/PI staining, cell proliferation by MTT methods, the expression of both Akt and p-Akt by immunocytochemistry, Rt-PCR and Western blot, glucose uptake by radiation isotope ^3H-G. Results 1. The Akt gene significantly decreased both AR and DR of MSCs under hypoxia （P〈0.01） and there was no significant differene （P〉0.05） between different durations. 2. The Akt gene ,significantly enhanced the capacity of proliferation of MSCs despite normoxic or hypoxic environment, but the capacity of proliferation of MSCs was significantly down-regulated in hypoxia than in nomoxia （P〈0.01） . 3. The Akt gene significantly increased the expression of Akt mRNA （P〈0.01） and protein （P〈0.01） in normoxia, and increased the expression of p-Akt protein （P〈0.01） in hypoxia but did not change its expression in normoxia （P〉0.05） . Despite Akt gene transfection, both Akt and p-Akt protein were expressed in MSCs by immunocytochemistry while normoxia or hypoxia. 4. The Akt gene significantly enhanced the ^3 H-G uptake ratio of MSCs at normoxia and hypoxia （P〈0. 01）; however, the ^3H-G uptake ratio significantly decreased in hypoxia than in normoxia （P〈0.01） . There was a significant negativecor relation between cell apoptosis and ^3H-G uptake （r=-1. 47, P〈0.01） , but a significant positive correlation between cell proliferation and ^3H-G uptake （r=0.79, P〈0.01） . Conclusion Akt gene transfection may significantly enhance the capacity of MSCs to endure hypoxia, and the capacity may be related