[Modification and activity observation of hepatocyte growth factor gene on bone marrow mesenchymal stem cells].
OBJECTIVE
To evaluate the transfection efficiency and expression level of hepatocyte growth factor (HGF) by transfecting a recombinant adenovirus carrying HGF gene (Ad-HGF) into bone marrow mesenchymal stem cells (BMSCs) and to explore the effect of the expression supernatant on BMSCs in vitro so as to lay a foundation for the manufacture of gene medicine which expresses efficient cell factors.


METHODS
Rat BMSCs were isolated using Percoll density gradient method and cultured according to the adherent property of BMSCs. The expression of c-Met was detected by immunohistochemical examination. BMSCs were infected with a recombinant adenovirus carrying green fluorescent protein gene (Ad-GFP) at multiplicity of infection (MOI, 0, 25, 50, 100, and 200 pfu/cell). To select an optimal MOI, the transfection efficiency and the degree of cell damage were assayed by flow cytometry and MTT, respectively, at 48 hours after transfecting. The expression of HGF in BMSCs transfected with optimal MOI Ad-HGF was measured with ELISA assay. MTT method was used to evaluate the proliferation effect of HGF expression supernatant on BMSCs.


RESULTS
Immunohistochemical staining showed that BMSCs expressed c-Met receptor for HGF. At 48 hours after transfecting with different MOI Ad-GFP (0, 25, 50, 100, and 200 pfu/cell), the transfection efficiencies were 0.34% +/- 0.04%, 40.72% +/- 0.81%, 61.72% +/- 1.04%, 85.33% +/- 0.83%, and 17.91% +/- 0.63%, respectively; and the highest transfection efficiency was observed at 100 pfu/cell MOI. The cell damage was obviously observed when MOI was 200 pfu/cell. The expression of HGF in BMSCs reached the highest level after being transfected with 100 pfu/cell MOI Ad-HGF for 48 hours. The expression product could stimulate the proliferation of BMSCs. The proliferation of BMSCs gradually rose with the increase of HGF protein, and reached the highest level at 10% (320 pg).


CONCLUSIONS
BMSCs can be transfected efficiently with Ad-HGF and express HGF protein, which stimulates the proliferation of BMSCs. It suggests that BMSCs is an ideal repair cells with gene vector.
Ha
Xiaoqin
X
Medical Experiment Center, Stem Cells and Gene Medicine Key Laboratory of Gansu Province, Lanzhou General Hospital, Lanzhou Command of Chinese PLA, Lanzhou Gansu 730050, PR China. haxq@yahoo.com
Dong
Fang
F
Lv
Tongde
T
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D005822:Genetic Vectors / Q000235:genetics; D017228:Hepatocyte Growth Factor / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection
D017228:Hepatocyte Growth Factor