摘要： 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 marrow stromal 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。
关键词:
低氧诱导因子1&alpha,
；慢病毒载体；骨髓间充质干细胞
Abstract: Objective: The study was designed to construct Lenti-HIF-1α, and detect HIF-1α expression and location in bone marrow mesenchymal stem cells (BMSCs) transduced by Lenti-HIF-1α. Methods: According to human HIF-1α gene sequence (NM_001530), its primer was designed and was amplified through PCR. The eukaryotic expression vector (pEGFP-N1-HIF-1α) 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-1α (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-1α. The analysis of target gene expression was done with qPCR. Besides, the immunohistochemistry examination was also completed to observe the location of HIF-1α 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-1α 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-1α 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-1α, and target gene located in the nucleus of BMSCs. This study will lay the foundation for experimental studies of bone defect repair using HIF-1α-mediated BMSCs in the future.
Key words:
hypoxia inducible factor 1α,
lentiviral vector,
bone marrow mesenchymal stem cells
中图分类号:
王瀛，张凯，邹多宏，何家才，王元银，周健. 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[J]. 《口腔颌面外科杂志》, 2012, 22(5): 323-327.
WANG Ying, ZHANG Kai, ZOU Duo-hong, HE Jia-cai, WANG Yuan-yin, ZHOU Jian. Construction of Lentiviral Vector Carrying HIF-1α and Detection of the Target Gene Expression in BMSCs[J]. 《Journal of Oral and Maxillofacial Surgery》, 2012, 22(5): 323-327.