[Research of lentiviral vector mediated growth factor gene-modified bone marrow mesenchymal stem cells].
OBJECTIVE
To construct lentiviral vector carrying the human hepatocyte growth factor (hHGF) gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells (BMSCs) by infecting the BMSCs.


METHODS
The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine 2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multiplicities of infection (MOI) was identified with fluorescent microscope, the efficiency of infection with flow cytometry (FCM) analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR.


RESULTS
Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 x 10(8) TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene.


CONCLUSIONS
By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.
Zhu
Jinhai
J
Department of Hepatobiliary Surgery, Affiliated Union Hospital, Fujian Medical University, Fuzhou Fujian 350001, PR China. zhujinhai@tom.com
Chen
Yanling
Y
D000818:Animals; D005822:Genetic Vectors; D017228:Hepatocyte Growth Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism* / Q000821:virology*; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
C066855:HGF protein, human; D017228:Hepatocyte Growth Factor