低氧诱导因子-1α慢病毒载体的构建及其在骨髓基质干细胞内表达的检测

Construction of Lentiviral Vector Carrying HIF-1α and Detection of the Target Gene Expression in BMSCs

目的:构建低氧诱导因子-1α(HIF-1α)的慢病毒载体(Lenti-HIF-1α),转染骨髓基质干细胞(bone marrowstromal cells,BMSCs)后,检测HIF-1α在BMSCs内的表达,并鉴定其位置。方法:根据人的HIF-1α基因(NM_001530)序列行引物设计及序列片段的PCR扩增,将目的基因PCR产物连接到载体pEGFP-N1上,构建真核表达载体pEGFP-N1-HIF-1α。将目的基因PCR产物和目的载体用NheI和BamHI分别进行酶切,对质粒进行鉴定。采用LR重组系统将目的序列重组到慢病毒载体plenti6.3V5-DEST上,构建慢病毒载体Lenti-HIF-1α(Lenti-LacZ为对照组)。检测慢病毒滴度后,转染BMSCs,检测目的基因的表达。通过细胞免疫组织化学法观察HIF-1α在BMSCs内的位置。结果:通过对构建质粒克隆进行测序及酶切,证实真核表达载体pEGFP-N1-HIF-1α构建成功。用Lenti-HIF-1α转染BMSCs细胞,0、1、4、7、14和21 d后qPCR检测。结果表明HIF-1α在转染后的第4天开始有明显的过表达,且持续到第21天。细胞免疫组织化学结果显示目的基因位于BMSCs的核内。结论:成功构建了慢病毒载体Lenti-HIF-1α,且转染的目的基因位于BMSCs细胞核内,为HIF-1α介导的BMSCs进行体内实验研究奠定了基础。

Objective： The study was designed to construct Lenti-HIF-lα, and detect HIF-lα expression and location in bone marrow mesenchymal stem ceils （BMSCs） transduced by Lenti-HIF-lα. Methods： According to human HIF-lα gene sequence （NM_O01530）, its primer was designed and was amplified through PCR. The eukaryotic expression vector （pEGFP-N1-HIF-lα） was constructed by connecting the PCR products of the target gene to the vector pEGFP-N1. To identify the plasmid, target gene PCR product and the purpose vector were digested by NheI and BamHI. Lenti-HIF-lct （control group, Lenti-LacZ） was constructed using the LR recombination system （the lentiviral vector plenti6.3V5-DEST）. After lentiviral titer was detected, BMSCs was transduced by Lenti-HIF-lα. The analysis of target gene expression was done with qPCR. Besides, the immunohistochemistry examination was also completed to observe the location of HIF-lα in BMSCs. Results： The results of plasmid sequencing and digestion confirmed that the eukaryotic expression vector pEGFP- N1-HIF-1α was successfully constructed. After Lenti-HIF-lα was transduced to BMSCs at 0 d, 1 d, 4 d, 7 d, 14 d and 21 d, the results of qPCR showed that the over-expression of HIF-lα was detected on 4 d, and continued until the 21 d. Immunohistochemical results showed that the target gene was located in the nucleus of BMSCs. Conclusion： We successfully constructed the Lenti-HIF-lα, and target gene located in the nucleus of BMSCs. This study will lay the foundation for experimental studies of bone defect repair using HIF-lα-mediated BMSCs in the future.