大鼠骨髓间充质干细胞经低氧诱导因子-1α基因转染后的生物学特性

Biological characteristics of rat bone marrow mcsenchymal stem cells transfected with hypoxia-inducible factor-let gene

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-la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Westernblot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G，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，G．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基因成功转染人大鼠BMSCs。

Objective To evaluate the biological characteristics of rat bone marrow mesenchymal stem cells （BMSCs） transfected with hypoxia-inducible factor-1α（HIF-1 α） gene. Metllods 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 trans- fection of HIF-1α gene was confirmed by immuno-cytochemistry. Simple BMSCs and BMSCs-pcDNA3.1 cells were used as control ceils. 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 MTr 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-la ceils 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 ceils/the total number of ceils examined） decreased in BMSC-HIF-1α cells compared with the control cells. The proportion of ceils 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 ceils. The number of BMSCs-HIF-1α 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.