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目的:构建Hif-1α基因(野生型、点突变型)慢病毒真核表达载体,并研究其对兔BMSCs(骨髓间充质干细胞)成骨基因表达的影响。方法:根据野生型人源Hif-1α基因序列和确定酶切位点的点突变型序列及质粒抽提、PCR等制备Hif-1α基因(野生型、点突变型)慢病毒液;转染兔BMSCs后通过免疫荧光显微镜及qPCR定量分析等检测被转染细胞目的基因Hif-1α和BMP-2的表达。结果:免疫荧光显微镜下可见,转染7d后野生型组和点突变组细胞均未见明显荧光,转染14d后两组细胞均呈现较明显的绿色荧光;qPCR定量结果显示,转染7d后即有Hif-1α和BMP-2基因的显著表达并在转染14d后仍然显示较高表达水平。讨论:免疫荧光显微镜和qPCR定量分别从定性和定量两个方面确定了转染细胞目的基因表达。结论:通过野生型人源Hif-1α基因序列信息和确定酶切位点的点突变型序列信息可以构建野生型和点突变型Hif-1α基因慢病毒真核表达载体;Hif-1α基因慢病毒转染可以促进目的细胞成骨基因表达。
OBJECTIVE: To construct the eukaryotic expression vector of Hif-1α gene (wild-type and point mutant) lentivirus and study its effect on the osteogenic gene expression of BMSCs (bone marrow mesenchymal stem cells) in rabbits. Methods: Hif-1α gene (wild type and point mutation) lentivirus was prepared according to wild-type human Hif-1α gene sequence, point mutation sequence and plasmid extraction and PCR. The expression of Hif-1α and BMP-2 in transfected cells was detected by immunofluorescence microscopy and qPCR quantitative analysis after BMSCs. Results: Immunofluorescence microscopy showed that no significant fluorescence was observed in the wild-type group and the point-mutated group 7 days after transfection, and both cells showed more obvious green fluorescence after 14 days of transfection. Quantitative qPCR results showed that after 7 days of transfection, That is, there was significant expression of Hif-1α and BMP-2 gene and still showed higher expression levels after 14 days of transfection. Discussion: Quantitative immunofluorescence microscopy and qPCR determine the expression of genes of interest in transfected cells qualitatively and quantitatively, respectively. Conclusion: Hif-1α lentivirus eukaryotic expression vector of wild-type and point mutant Hif-1α gene can be constructed by sequence information of wild-type Hif-1α gene and point mutation sequence information of restriction enzyme site. Hif-1α lentivirus Transfection can promote the osteoblast gene expression in the target cells.