摘要
背景:成骨作用和血管发生在骨的形成和修复过程中紧密结合,而低氧诱导因子1α被认为是促血管新生相关基因调控中最重要的核心转录因子,其可促进缺氧部位血管的形成,进而促进骨的形成。目的:构建野生型和点突变型两个低氧诱导因子1α慢病毒真核表达载体Lenti-HIF1α-IRES-EGFP,并比较其对兔骨髓间充质干细胞成骨基因表达的影响。方法:首先根据野生型人源低氧诱导因子1α基因序列信息和确定酶切位点的点突变型序列信息构建低氧诱导因子1α基因野生型和点突变型两个慢病毒真核表达载体;然后采用制备的病毒液转染兔骨髓间充质干细胞。结果与结论:免疫荧光显微镜观察显示,转染7 d后野生型组和点突变型组细胞均未见明显荧光,转染14 d后两组细胞均呈现明显的绿色荧光;qPCR定量分析结果表明,转染7 d后即有低氧诱导因子1α和骨形态发生蛋白2基因的显著表达,转染14 d后,两基因仍然显示较高的表达水平。说明实验成功构建野生型和点突变型低氧诱导因子1α基因慢病毒真核表达载体,转染后可以促进兔骨髓间充质干细胞成骨基因的表达。
BACKGROUND：Osteogenesis is closely integrated with angiogenesis in bone formation and repair process, and hypoxia-inducible factor 1 alpha （HIF-1α） is considered to be the most important core transcription factor promoting angiogenesis gene regulation, which may promote the formation of blood vessels at hypoxia portion, and thus contribute to bone formation. OBJECTIVE：To construct the Lenti-HIF-1α-IRES-EGFP （wild type） and Lenti-HIF-1α-IRES-EGFP （point mutant type） lentiviral eukaryotic expression vectors and to detect their impact on the osteogenic gene expression of rabbit bone marrow mesenchymal stem cells. METHODS：The Lenti-HIF-1α-IRES-EGFP （wild type） and Lenti-HIF-1α-IRES-EGFP （point mutant type） lentiviral eukaryotic expression vectors were constructed according to the wild type human HIF-1αgene sequence and the determined restriction sites of human HIF-1αpoint mutant sequence. Rabbit bone marrow mesenchymal stem cells were transfected with the prepared Lenti-HIF-1α-IRES-EGFP （wild type） and Lenti-HIF-1α-IRES-EGFP （point mutant type） virus solution. RESULTS AND CONCLUSION：Immunofluorescence microscopy observations indicated that the cells of Lenti-HIF-1α-IRES-EGFP （wild type） group and Lenti-HIF-1α-IRES-EGFP （point mutant type） group showed no obvious fluorescence on transfected 7 days, and two groups of cells showed a more obvious green fluorescent after transfection 14 days. Quantitative PCR analysis results showed that there were obvious HIF-1αand bonemorphogenetic protein 2 gene expressions on days 7 after transfection and the two genes stil showed highly expression levels after transfection 14 days. The two lentiviral eukaryotic expression vectors of Lenti-HIF-1α-IRES-EGFP （wild type） and Lenti-HIF-1α-IRES-EGFP （point mutant type） could be constructed according to the wild type human HIF-1αgene sequence and the determined restriction sites of human HIF-1αpoint mutant sequence;HIF-1αgene can promote the osteogenic gene expression of lentivirus-transfected rabbit bone marrow mesenchymal stem cells.
出处
《中国组织工程研究》
CAS
CSCD
2014年第1期57-62,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金重点项目(81070806)
浙江省自然科学基金(LQ12H14002)
上海市科委医学重点课题(10JC1413300)
杭州市医药卫生科技计划项目(2012A015)~~
