缺氧诱导因子-1α基因转染人间充质干细胞的体内促血管生成作用  

Effect of hypoxia-inducible factor 1αgene transfected into human mesenchymal stem cells on angiogenesis in vivo

作　　者：胡鸢[1] 段小军[1] 杨柳[1] 

机构地区：[1]第三军医大学西南医院关节外科中心,重庆400038

出　　处：《中华实验外科杂志》2006年第8期993-996,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(30270375)

摘　　要：目的研究将缺氧诱导因子-1α(HIF-1α)基因转染人间充质干细胞(hMSCs)的体内促血管生成作用,为促进组织工程骨血管化提供新的方法和策略。方法构建pIRES3/HIF-1α逆转录病毒载体,制备含目的基因的重组逆转录病毒,感染hMSCs。用逆转录-聚合酶链反应(RT- PCR)法检测转基因hMSCs表达HIF-1α的水平,用免疫组织化学法检测转基因hMSCs在缺氧和常氧条件下HIF-1α蛋白的存在情况。体内促血管生成的实验分为两组：实验组采用转基因hMSCs．对照组采用未转基因的hMSCs。两种细胞分别接种到13d的鸡胚绒毛尿囊膜(CAM),3d后将CAM制片,在显微镜下观察并摄像。用图像分析方法测量并分析血管面积。结果转基因hMSCs中HIF-1αmRNA表达较未转基因的hMSCs明显增高。转染HIF-1α基因的hMSCs,缺氧条件下细胞核内HIF-1α能稳定存在；常氧条件下细胞核内HIF-1α不能稳定存在。图像分析结果显示实验组CAM较对照组CAM血管面积明显增多,差异有统计学意义(P＜0.01)。结论HIF-1α基因能稳定转染到hMSCs并得到强制表达。转染HIF-1α基因的hMSCs有明显的促血管生成作用,对于促进组织工程骨的血管化有重要作用。Objective To evaluate the effect of hypoxia-inducible factor 1α （HIF-1α）gene transfected into human mesenchymal stem cells （hMSCs） on angiogenesis in vivo. Thereby, provide a novel method and strategy for facilitating angiogenesis of tissue engineered bone. Methods Recombinant retrovirus vector of HIF-1α gene was constructed and transfected into packaging cell PT67. The virus supernatant from infected PT67 cell culture was used to infect hMSCs. Transgene expression in infected hMSCs was detected by RT-PCR analysis, and HIF-1α protein was detected under hypoxic conditions and normoxic conditions respectively by immunohistochemistry. On day 13 of chick embryo incubation, transgenic hMSCs and nontransgenic controls were implanted onto the surface of the CAM respectively. After 3 days, CAMs were photographed in ovo under a stereomicroscope and then fixed in Bouin＇s fluid, dehydrated in ethanol, embedded in resin, mounted on glass slides. Specimens were photographed and capillary areas were measured and analysed using Image-Pro Plus 4.5, data were analyzed for statistical significance using t-test. Results HIF-1α mRNA expression in transgenic hMSCs increased remarkably compared with nontransgenic controls in hypoxia. Cell nucleus that stained positively for HIF-1α were observed in transgenic hMSCs culturing under hypoxic conditions but no in those culturing under normoxic conditions. Image analysis revealed a statistically significant increase in capillary areas in transgenic hMSCs compared with nontransgenic controls （ P 〈 0.01 ）. Conclusion HIF-1α genes have been transfected into hMSCs and expressed effectively. Transgenic hMSCs appeared to significant induction of angiogenesis compared with nontransgenic controls. HIF-1α overexpression may represent a promising therapeutic strategy for promoting neovascularization in tissue engineered bone.

关 键 词：缺氧诱导因子-1 间充质干细胞 血管生成 

分 类 号：R318.0[医药卫生—生物医学工程]