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  

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

作　　者：徐宠俊 何荷蕃[1] 林群[2] 章涛[3] XU Chongjun;HE Hefan;LIN Qun;ZHANG Tao(Department of Anesthesiology,Second Affiliated Hospital,Fujian Mdeical University,Quanzhou 362000,China;Department of Anesthesiology,First Affiliated Hospital,Fujian Mdeical University,Fuzhou 350005,China;Department of Immunology,Fujian Mdeical University,Fuzhou 350004,China)

机构地区：[1]福建医科大学附属第二医院麻醉科,福建泉州362000 [2]福建医科大学附属第一医院麻醉科,福建福州350005 [3]福建医科大学免疫实验室,福建福州350004

出　　处：《吉林大学学报（医学版）》2021年第1期16-24,共9页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(30972972)。

摘　　要：目的:构建人肝细胞生长因子(HGF)基因慢病毒载体pNL-HGF-增强绿色荧光蛋白(EGFP)并转染骨髓间充质干细胞(BMSCs),检测HGF基因在BMSCs中的表达。方法:采用基因重组技术,利用限制性内切酶酶切获取HGF基因序列并克隆到慢病毒载体pNL-EGFP上,构建重组慢病毒质粒pNL-HGF-EGFP;将慢病毒载体质粒pNL-EGFP和重组慢病毒质粒pNL-HGF-EGFP分别与慢病毒辅助包装质粒共转染人胚肾293T细胞,包装慢病毒并测定慢病毒滴度。将感染pNLEGFP的BMSCs作为对照组,感染pNL-HGF-EGFP的BMSCs作为实验组。RT-PCR法和ELISA法检测对照组和实验组BMSCs中HGF mRNA表达水平和蛋白水平。结果:酶切电泳和测序检测,重组慢病毒质粒的目的基因序列与GenBank公布的HGF基因序列完全一致。对照组包装的慢病毒滴度为1.2×10^7 TU·mL^-1,实验组慢病毒滴度为1.5×10^6 TU·mL^-1;RT-PCR法检测,实验组BMSCs中HGF mRNA表达水平较对照组明显升高(P<0.05);ELIAS法检测,实验组BMSCs上清中HGF蛋白水平较对照组明显升高(P<0.05)。结论:成功构建了pNL-HGF-EGFP慢病毒载体,且BMSCs中HGF mRNA表达水平和蛋白水平明显升高。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 1.2×10^7 TU·mL^-1,and the lentivirus titer in experimental group was 1.5×10^6 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.

关 键 词：慢病毒 肝细胞生长因子 骨髓间充质干细胞 神经病理性疼痛 

分 类 号：Q782[生物学—分子生物学]