【摘要】 背景:作为种子细胞来源的骨髓间充质干细胞移植后能否在相对缺氧的体内环境下生存,是移植成功与否的重要环节。因此研究体外低氧环境下骨髓间充质干细胞的生长状态,可以为体内细胞移植提供实验依据。目的:观察体外低氧环境对大鼠骨髓间充质干细胞生长及血管内皮生长因子表达的影响。方法:分离培养大鼠骨髓间充质干细胞,光镜观察细胞的形态;取第3代骨髓间充质干细胞,按氧浓度分为两组,分别在常氧条件下(体积分数为21%氧气)和低氧条件下(体积分数为3%氧气)培养72 h。用CCK-8法和流式细胞术检测两组细胞的增殖情况,Western-blot印迹法检测常氧组和低氧组细胞中缺氧诱导因子1α与血管内皮生长因子蛋白的表达。结果与结论:①成功分离和培养了骨髓间充质干细胞,光镜下细胞呈长梭形,形态均一。②CCK-8法结果显示各时间点低氧组的细胞数目均高于常氧组,在36,48 h时活力增加显著(P<0.05)。③流式细胞术结果显示,与常氧组比较,低氧组处于S期的细胞数量增加,且细胞增殖指数也显著增加(P<0.05)。④Western-blot印迹法结果显示,常氧组细胞缺氧诱导因子1α及血管内皮生长因子蛋白仅有少量的表达,而低氧组两蛋白表达量均呈时间依赖性上调(P<0.05)。以上结果说明低氧可诱导体外培养的大鼠骨髓间充质干细胞的增殖,又上调了缺氧诱导因子1α与血管内皮生长因子蛋白的表达,随着低氧时间的延长呈升高趋势。更多还原

【Abstract】 BACKGROUND: Whether transplanted bone marrow mesenchymal stem cells under hypoxic conditions can survive is crucial for the successful cell transplantation. Therefore, studies on the growth of bone marrow mesenchymal stem cells under hypoxic conditions in vitro can provide experimental evidence for in vivo cell transplantation. OBJECTIVE: To observe the expression of vascular endothelial growth factor in bone marrow mesenchymal stem cells under hypoxia. METHODS: Rat bone marrow mesenchymal stem cells were obtained and cultured, and observed under light microscopy. Passage 3 cells were cultured under normoxia(21% O2) and hypoxia(3% O2), respectively, for 72 hours. Then cell counting kit-8 assay and flow cytometry were employed to detect cell proliferation in the two groups. Western blot assay was adopted to detect the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in the two groups. RESULTS AND CONCLUSION:(1)Rat bone marrow mesenchymal stem cells were obtained and cultured successfully, which were fusiform cells and had uniform shape under the light microscope.(2)The results of cell counting kit-8 assay showed that the number of cells in the hypoxic group was higher than that in the normoxic group at each time point, and cell viability increased significantly at hours 36 and 48(P < 0.05).(3)The results of flow cytometry demonstrated that the proportion of cells in S phase and cell proliferation index in the hypoxic group were significantly increased, compared with the normoxic group(P < 0.05).(4)Western blot results showedthat there was a small amount of the expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in the normoxic group, but the expression of these two proteins in the hypoxic group was increased in a time-dependent manner(P < 0.05). These findings suggest that hypoxia can induce proliferation of rat bone marrow mesenchymal stem cells cultured in vitro, and also raise hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a time-dependent manner.更多还原