摘要
背景:有研究表明骨髓间充质干细胞的生理环境氧体积分数低于常规培养用的20%-21%,适度低氧体积分数下,骨髓间充质干细胞表现出促进生长增殖,保持分化潜能的特性。目的:观察低氧预处理后的大鼠骨髓间充质干细胞的生物学特性,以期为低氧预处理后的骨髓间充质干细胞体内移植治疗寻找体外依据。方法:将大鼠骨髓间充质干细胞传代接种后置于体积分数1%O2,5%CO2的低氧培养箱内培养,以体积分数20%O2,5%CO2常氧培养的骨髓间充质干细胞同样封闭作为对照。结果与结论:MTT检测显示,在低氧条件下培养的经低氧预处理的骨髓间充质干细胞较常氧条件下培养的细胞在贴壁后会经历一个相对较长时间的潜伏期,随后才进入指数生长期,体积分数1%O2为非致死性的培养条件。锥虫蓝染色显示,封闭无血清条件下,两组细胞存活率均随着时间的推移而降低(P<0.05),而常氧组下降更快。流式细胞仪检测显示,低氧组的细胞表面分子标记物CD29(+),CD90(+),CD31(-),CD45(-)的表达与低氧预处理前差异无显著性意义,未向血管内皮细胞分化。实时定量PCR和酶联免疫实验检测结果显示,低氧预处理48 h后,骨髓间充质干细胞中血管内皮生长因子mRNA和蛋白的表达均升高(P<0.05)。Western-blot检测显示,低氧预处理24-48 h后,骨髓间充质干细胞中缺氧诱导因子1α的蛋白表达明显增多(P<0.05)。证实,体积分数1%O2能短暂抑制骨髓间充质干细胞的增殖,但不会产生致死性效应,可以作为低氧预处理的氧体积分数。体积分数1%O2预处理48 h后,骨髓间充质干细胞的表型未发生明显变化,仍维持其干性。低氧预处理提高骨髓间充质干细胞对缺血缺氧的耐受能力,机制可能与低氧诱导因子1α表达增多有关。
BACKGROUND:According to the previous studies, it is known that bone marrow mesenchymal stem cells live in the physiological environment of lower oxygen concentration compared with the common culture concentration (20%-21%). Hypoxia can promote bone marrow mesenchymal stem cells proliferation and maintain the characteristics of differentiation potential.
OBJECTIVE:To understand the biological characteristics of bone marrow mesenchymal stem cells after hypoxic preconditioning, and search for in vitro basis for bone marrow mesenchymal stem cells therapy in vivo with hypoxic preconditioning strategy.
METHODS:After passage and inoculation, bone marrow mesenchymal stem cells were cultured in the hypoxic incubator (1%O2, 5%CO2). Bone marrow mesenchymal stem cells cultured in the normoxic incubator (20%O2, 5%CO2) served as controls.
RESULTS AND CONCLUSION:Compared with bone marrow mesenchymal stem cells cultured under normoxia (20%O2), bone marrow mesenchymal stem cells cultured under hypoxia (1%O2) began to display exponential growth phase after a longer incubation period. Hypoxia (1%O2) was not the lethal condition for bone marrow mesenchymal stem cells. cellsurvival rates of both groups reduced over time, while those of bone marrow mesenchymal stem cells cultured under normoxia reduced faster (P〈0.05). Surface markers expression of bone marrow mesenchymal stem cells, including CD29(+), CD90(+), CD31(-), CD45(-), after hypoxic preconditioning did not change greatly when compared with the results before hypoxic preconditioning. The hypoxia preconditioned bone marrow mesenchymal stem cells did not differentiate into vascular endothelial cells. Hypoxia-inducible factor-1αprotein expression was promoted after 24 hours to 48 hours hypoxic preconditioning, and there was significant difference between the two time points (P〈0.05). Vascular endothelial growth factor expression increased at both transcriptional and protein levels after 48 hours hypoxic preconditioning (P〈0.05). Culture under hypoxia (1%O2) does not produce a lethal effect on bone marrow mesenchymal stem cells and can be used for hypoxic preconditioning. Surface markers of bone marrow mesenchymal stem cells after 48 hours hypoxic preconditioning exhibit no great changes and the hypoxia preconditioned bone marrow mesenchymal stem cells maintain the characteristics of stemness. Hypoxic preconditioning can promote the ability of bone marrow mesenchymal stem cells to survive under hypoxia/ischemic condition. The activation of the hypoxic response signal transduction pathway through hypoxia-inducible factor-1αmay explain this.
出处
《中国组织工程研究》
CAS
CSCD
2013年第49期8474-8480,共7页
Chinese Journal of Tissue Engineering Research
基金
the National Natural Science Foundation of China for the Youth,No.30900309~~