人肝细胞生长因子基因慢病毒载体的构建及其在骨髓间充质干细胞中的表达

doi:10.13481/j.1671-587x.20210103

Abstract

Abstract: Objective

To construct the human hepatocyte growth factor （HGF） gene lentivirus vector pNL-HGF-EGFP and transfect the bone marrow mesenchymal stem cells（BMSCs），and to detect the expression of HGF gene in the BMSCs.

Methods

The recombinant lentivirus plasmid pNL-HGF-EGFP was constructed by using restriction enzyme digestion to obtain HGF gene sequence and cloned into the lentivirus vector pNL-EGFP.The lentivirus vector plasmid pNL-EGFP and recombinant lentivirus plasmid pNL-HGF-EGFP were co-transfected into the 293T cells with lentivirus assisted packaging plasmid， respectively. The lentivirus was packaged and the titer of lentivirus was determined.The BMSCs infected with pNL-EGFP were used as control group， and the BMSCs infected with pNL-HGF-EGFP were used as experimental group.RT-PCR and ELISA methods were used to detect the expression levels of HGF mRNA and the levels of HEG protein in the BMSCs in control group and experimental group.

Results

The electrophoresis and sequencing results showed that the target gene sequence of the recombinant lentivirus plasmid was consistent with the HGF gene sequence published in GenBank.The packaged lentivirus titer in control group was 1.2×10⁷ TU·mL^－1， and the lentivirus titer in experimental group was 1.5×10⁶ TU·mL^－1.The RT-PCR detection results showed that the expression level of HGF mRNA in the BMSCs in experimental group was higher than that in control group（P<0.05）.The ELIAS results showed that the level of HGF protein in the BMSCs in experimental group was significantly higher than that in control group （P<0.05）.

Conclusion

The lentiviral vector pNL-HGF-EGFP is successfully constructed， and the expression level of HGF mRNA and and the level of HGF protein in the BMSCs are significantly increased.

Key words: lentivirus, hepatocyte growth factor, bone marrow mesenchymal stem cell, neuropathic pain

CLC Number:

Q782

Chongjun XU,Hefan HE,Qun LIN,Tao ZHANG. Construction of human hepatocyte growth factor gene lentivirus vector and its expression in bone marrow mesenchymal stem cells[J].Journal of Jilin University(Medicine Edition), 2021, 47(1): 16-24.

References 17

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Construction of human hepatocyte growth factor gene lentivirus vector and its expression in bone marrow mesenchymal stem cells

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