[Adenoviral vector-mediated human hepatocyte growth factor gene transfection for its high expression in rabbit bone marrow-derived mesenchymal stem cells].
OBJECTIVE
To obtain high expression of human hepatocyte growth factor (HGF) in passaged rabbit bone marrow-derived mesenchymal stem cells (BMSCs) via recombinant adenovirus vector mediated HGF gene transfection, and explore the feasibility of this strategy for local treatment of avascular necrosis of the femoral head (ANFH).


METHODS
HGF gene was subcloned into the adenovirus shuttle plasmid pDC316, and the products were co-transfected into HEK293 cells with the helper plasmid pBHGlox deltaE1,3Cre. The recombinant adenovirus Ad-HGF was generated by homologous recombination of the 2 plasmids in HEK293 cells. After PCR identification, Ad-HGF was amplified and purified and its titer measured by TCID50 assay before transfected into the second passage of rabbit BMSCs. The transcription and expression of HGF gene in the transfected BMSCs was detected by RT-PCR, in situ hybridization, and immunological histochemistry.


RESULTS
Ad-HGF was successfully constructed with a titer of 2.6x10(10) TCID50/ml. The expressions of HGF mRNA and protein were confirmed in the transfected BMSCs.


CONCLUSIONS
Ad-HGF transfection allows efficient expression of HGF protein in rabbit BMSCs, and this success may facilitate further study of local treatment of ANFH using HGF-expressing BMSCs.
Wen
Qian
Q
Institute of Molecular Immunology, School of Biotechnology, Department of Orthopedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. wencaoxi@163.com
Ma
Li
L
Jin
Dan
D
Cui
Jian-de
JD
Luo
Wei
W
Wang
Xiao-ning
XN
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D011817:Rabbits; D014162:Transfection
C066855:HGF protein, human; D017228:Hepatocyte Growth Factor