Abstract:

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.

Key words: BMP-7, lentiviral vector, HSC-T6