Construction of recombinant adenoviruses carrying human hepatocyte growth factor and investigation of its effect on human fetal hepatocytes in vitro
- VernacularTitle:人肝细胞生长因子重组腺病毒的构建及其对人胎肝细胞细胞周期的影响
- Author:
Yong LIN
;
Weifen XIE
;
Zhongbing ZHANG
- Publication Type:Journal Article
- Keywords:
Hepatocyte growth factor;
Replication-deficient recombinant adenovirus;
Gene therapy;
Hepatocyte transplantation
- From:
Chinese Journal of Digestion
1998;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the replication deficient recombinant adenoviruses (AdHGF) of human hepatocyte growth factor (HGF) and green fluorescent protein (GFP) cDNA derived from CMV promoter using homologous recombination in bacteria provided by AdEasy system and to further investigate the effect of HGF on human fetal hepatocyte growth. Methods The HGF cDNA was obtained from the plasmid-pUCHGF by digestion, and the shuttle plasmid-pAdTrack-CMV-HGF in which the HGF cDNA was inserted into the downstream of CMV promoter was established by ligation. Then the linearized shuttle plasmid was co-transformed into bacteria with backbone vector AdEasy-1 to obtain the recombinant adenoviral plasmid-pAdHGF by homologous recombination. After packed in 293 cells, the recombinant adenoviruses-AdHGF was generated. The expression of HGF in human fetal hepatocyte was detected by RT-PCR and the effect of AdHGF on the cell cycle of fetal hepatocyte was examined by flow cytometry. Results The recombinant plasmid pAdHGF was established by homologous recombination and confirmed by restriction endonuclease digestion and sequencing. GFP expression could be observed on the third day after packing of the linearized pAdHGF in 293 cells and 4?10 10 efu/ml titer of AdHGF was obtained by CsCl gradient purification. Three days after the human fetal hepatocytes were infected by the viruses, expression of HGF in hepatocytes increased significantly and most of the hepatocytes in G 0/G 1 stages was changed to that in S and G 2/M stages.Conclusions AdHGF can be simply and rapidly generated using AdEasy system. The infection of human fetal hepatocytes by AdHGF could result in the high expression of HGF and promote the cell regeneration. AdHGF may serve as a new tool for hepatocyte transplantation and gene therapy of hepatic fibrosis.
