摘要
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1×104ifμ/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。
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（P0.01）.There was no significant difference between the empty vector control group and the blank group（P0.05）. Conclusion Lentiviral vector carrying BMP-7 gene has been successfully constructed and maintains high expression in HSC-T6 cells.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2010年第10期1189-1193,1216,共6页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(30971332)
上海市科委"浦江人才计划项目"(10PJ1407600)~~
