摘要目的 评价大鼠骨髓间充质干细胞(BMSCs)经低氧诱导因子-1α(HIF-1α)基因转染后的生物学特性.方法 将构建好的HIF-1α基因表达载体(pcDNA3.1-HIF-1α)运用电穿孔法转染入第3代BMSCs中得到稳定表达HIF-1α的BMSCs( BMSCs-HIF-1α细胞).以单纯BMSCs和电转染pcDNA3.1的BMSCs( BMSCs-pcDNA3.1细胞)作为对照.采用免疫细胞化学法鉴定HIF-1α基因表达载体是否转染成功；低氧培养后,采用Western blot法测定HIF-1α表达；流式细胞术对转染后细胞进行周期与凋亡的检测,记录G1期、G2期和S期细胞比例,计算增殖指数(PI)；MTT法绘制细胞生长曲线,记录细胞数目.结果 BMSCs-HIF-1α细胞核内充满蓝紫色深染颗粒,其余两种细胞未见深染颗粒.与BMSCs和BMSCs-pcDNA3.1细胞比较,BMSCs-HIF-1α细胞HIF-1α表达上调,细胞凋亡率降低,S期和G2期细胞比例和PI升高,G1期细胞比例降低,细胞数目增多(P＜0.05).BMSCs和BMSCs-pcDNA3.1细胞上述各指标比较差异无统计学意义(P＞0.05).结论 HIF-1α基因成功转染入大鼠BMSCs.

abstractsObjective To evaluate the biological characteristics of rat bone marrow mesenchymal stem cells (BMSCs) transfected with hypoxia-inducible factor-1α(HIF-1 α) gene.Methods The rat BMSCs of 3rd generation and the vector expressing HIF-1α gene (pcDNA3.1-HIF-1α) were provided by department of anesthesia,Tangdu Hospital,the 4th Military Medical University.BMSCs expressing HIF-1α gene (BMSCs-HIF-1α cells) were constructed by transfection of vector pcDNA3.1-HIF-1α into BMSCs by means of electroporation.Successful transfection of HIF-1α gene was confirmed by immuno-cytochemistry.Simple BMSCs and BMSCs-pcDNA3.1 cells were used as control cells.After being cultured in hypoxic condition HIF-1α expression was detected by Western blot analysis.Flow cytometry was used to determine the proportion of cells in G1,G2 and S phase and detect apoptosis.The proliferation index (PI) was calculated.The cell growth curve was described by MTT assay and the number of the 3 types of cells was recorded.Results A large number of deep blue granules were observed in the nuclei of BMSCs-HIF-1α cells using immuno-cytochemistry but no such granule was found in the two types of control cells.HIF-1α expression was significantly up-regulated and apoptosis rate (the number of apoptotic cella/the total number of cells examined) decreased in BMSC-HIF-1α cells compared with the control cells.The proportion of cells in S and G2 phase was significantly higher and the proportion of cells in G1 phase was significantly lower and PI higher in BMSCs-HIF-1α cells than in the control cells.The number of BMSCs-HIF-lα cells was significantly higher than the number of the two types of control cells at day 3-8 of culture.There was no significant difference in the above variables between BMSCs and BMSCs-pcDNA3.1 cells.Conclusion BMSCs-HIF-1α is successfully constructed by transfection of vector pcDNA3.1-HIF-1α gene into BMSCs by means of electroporation.