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目的探讨缺氧诱导因子-1α(hypoxia induced factor-1α,HIF-1α)基因转染是否提高缺氧间充质干细胞(mesenchymal stem cells,MSCs)的耐缺氧能力以及这种能力是否与增加葡萄糖摄取有关。方法将MSCs分为常氧非转染组(即对照组)、常氧转染组、缺氧非转染组和缺氧转染组,分别于常氧(5%CO2,37℃)和缺氧(94%N2、1%O2和5%CO2,37℃)条件下孵育8 h。免疫细胞化学、RT-PCR和Western blot法检测HIF-1α表达,流式细胞仪检测细胞凋亡率(AR)、死亡率(DR),MTT法检测细胞增殖,放射同位素法检测氚标葡萄糖(3H-G)摄取量。结果①与常氧非转染组相比,常氧转染组HIF-1αmRNA表达明显增高(P<0.01),但其蛋白表达2组间差异无统计学意义(P=0.187);与缺氧非转染组相比,缺氧转染组的HIF-1αmRNA和蛋白表达均明显增高(P<0.01)。②缺氧转染组MSCs的AR(P=0.001)和DR(P=0.003)均明显低于缺氧非转染组,但均明显高于常氧非转染组(P<0.01)。③与缺氧非转染组相比,缺氧转染组MSCs增殖(P=0.004)明显提高,但明显低于常氧非转染组(P=0.001)。④与缺氧非转染组相比,缺氧转染组MSCs对3H-G的摄取量明显提高(P=0.004),但明显低于常氧非转染组(P=0.001)。⑤AR与HIF-1α蛋白表达(r=-0.71,P=0.005)3、H-G摄取量(r=-0.65,P=0.004)均呈明显负相关,HIF-1α蛋白表达与3H-G摄取量呈明显正相关(r=0.77,P=0.003)。结论 HIF-1α基因可显著提高MSCs的耐缺氧能力,其机理可能与HIF-1α基因提高MSCs对葡萄糖的摄取有关。
Objective To investigate whether hypoxia-induced factor-1α (HIF-1α) gene transfection can enhance hypoxia tolerance of mesenchymal stem cells (MSCs) and whether this ability is associated with increased glucose Ingestion related. Methods MSCs were divided into normoxia non-transfection group (control group), normoxia transfection group, hypoxia non-transfection group and hypoxia transfection group, respectively, under normoxia (5% CO2, 37 ℃) Oxygen (94% N2, 1% O2 and 5% CO2, 37 ° C) for 8 h. Immunocytochemistry, RT-PCR and Western blot were used to detect the expression of HIF-1α. Flow cytometry was used to detect the apoptotic rate (AR) and mortality (DR). Cell proliferation was measured by MTT assay. Tritiated glucose 3H-G) uptake. Results ① The expression of HIF-1α mRNA in normoxia group was significantly higher than that in normoxia group (P <0.01), but there was no significant difference between the two groups (P = 0.187) Compared with non-transfected group, the expression of HIF-1αmRNA and protein in hypoxic group were significantly increased (P <0.01). ② The AR (P = 0.001) and DR (P = 0.003) of MSCs in hypoxia group were significantly lower than those in hypoxia and non-hypoxia group, but significantly higher than those in non-hypoxia group (P <0.01). ③Compared with hypoxia non-transfected group, the proliferation of MSCs in hypoxia-transfected group was significantly increased (P = 0.004), but significantly lower than that in non-transfected group (P = 0.001). ④ The uptake of 3H-G by MSCs in hypoxia group was significantly higher than that in hypoxia-non-transfected group (P = 0.004), but significantly lower than that in normoxic non-transfected group (P = 0.001). ⑤ There was a negative correlation between the expression of HIF-1αandAR and HIF-1α (r = -0.71, P = 0.005) 3 and HG uptake (r = -0.65, P = 0.004) There was a significant positive correlation (r = 0.77, P = 0.003). Conclusion The HIF-1α gene can significantly improve the hypoxia tolerance of MSCs. The mechanism may be related to the increase of MSCs glucose uptake by HIF-1α gene.