WANG Ying, ZHANG Kai, ZOU Duo-hong, HE Jia-cai, WANG Yuan-yin, ZHOU Jian
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.