大鼠BMP-7基因慢病毒载体构建及其在肝星状细胞中的表达

目的 构建携带大鼠BMP-7基因的慢病毒载体并实现该基因在肝星状细胞株HSC-T6中的稳定高表达，为进一步研究BMP-7的抗肝纤维化功能奠定基础。方法 从大鼠细胞基因中提取并用PCR方法扩增BMP-7目的基因，构建BMP-7重组表达载体Lenti-copGFP/puro-BMP7，脂质体法将重组慢病毒载体和包装质粒混合物共转染293TN工具细胞，包装产生慢病毒颗粒。慢病毒感染H1299细胞，根据细胞绿色荧光蛋白（GFP）的表达水平测定病毒滴度。将HSC-T6细胞分为空白组、空病毒对照组及BMP-7慢病毒感染组，分别用Real-Time PCR和Western blotting方法检测BMP-7 mRNA和蛋白的表达。结果 PCR扩增检测阳性菌落和测序证实，成功构建大鼠BMP-7基因重组慢病毒载体。倒置荧光显微镜下观察可见，H1299细胞呈绿色荧光，并测得病毒滴度为1×104 ifμ/μL。重组慢病毒感染HSC-T6后，Real-time PCR和Western blotting方法分别检测到BMP-7 mRNA和蛋白均稳定高表达。BMP-7慢病毒感染组与空病毒对照组比较，差异有统计学意义（P<0.01）；空病毒对照组与空白组比较，差异无统计学意义（P<0.05）。结论 成功构建了带有大鼠BMP-7基因的慢病毒载体，并实现其在HSC-T6的稳定高表达。

Construction of lentiviral vector carrying BMP-7 gene and its expression in hepatic stellate cells in rats

Objective To construct lentiviral vector carrying BMP-7 gene and maintain its high expression in hepatic stellate cell line (HSC-T6) in rats, and to lay the foundation for further research of BMP-7 anti-liver fibrosis. Methods The BMP-7 was extracted and amplified by PCR and constructed into Lenti-copGFP/puro-BMP7. The 293TN cells were contransfected with the recombinant lentiviral vector together with lentivirus package plasmid to produce lentiviral particles. H1299 cells were infected with Lv-BMP7. Virus titer was measured according to the expression level of GFP expressed in H1299 cells. The HSC-T6 cells were divided into blank group, empty vector control group, and experimental group. The mRNA expression of BMP-7 in HSC-T6 was detected with Real-Time PCR. The protein expression of BMP-7 was observed with Western blotting method. Results The recombinant pLV-BMP-7 vector was confirmed by restriction endonuclease analysis and DNA sequencing. H1299 cells were observed in green fluorescence, and virus titer reached to 1×104 ifμ/μL. In infected HSC-T6 cells the high expressions of BMP-7 mRNA and protein were confirmed. The difference between the experimental group and the blank group was significant (P<0.01). There was no significant difference between the empty vector control group and the blank group (P>0.05). Conclusion Lentiviral vector carrying BMP-7 gene has been successfully constructed and maintains high expression in HSC-T6 cells.