OBJECTIVETo construct a retroviral vector carrying human vascular endothelial growth factor (hVEGF (121)) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.

METHODShVEGF(121) cDNA was obtained from the plasmid pCDI/VEGF(121) and cloned into retroviral plasmid pLXSN. Recombinant plasmid was transferred to the retro virus packaging cell, PT-67, by lipofectamine mediated gene transfer. Mouse bone marrow stromal cells (MSCs) were transfected by the retrovirus. The integration of the hVEGF(121) cDNA into MSC genomic DNA and expression of the VEGF gene was detected. Proliferation assays of human umbilical vein endothelial cells (HUVECs) by VEGF(121) in culture medium were performed.

RESULTSRecombinant pLXSN/VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. hVEGF(121) gene was integrated into MSC genomic DNA after transfection, and the VEGF(121) protein was expressed. Proliferation assays showed VEGF(121) in culture medium was a biologically active protein and had a mitogenic effect on HUVEC.

CONCLUSIONSRecombinant retroviral vector carrying hVEGF(121) cDNA was successfully constructed. VEGF (121) protein expressed by MSCs had mitogenic effect biologically. This provides a further foundation for VEGF gene therapy for bone ischemic disease and bone tissue engineering.

Animals ; Bone Marrow Cells ; metabolism ; Cell Division ; DNA, Complementary ; genetics ; Endothelial Growth Factors ; genetics ; Endothelium, Vascular ; cytology ; Genetic Therapy ; Humans ; Lymphokines ; genetics ; Mice ; Plasmids ; Retroviridae ; genetics ; Stromal Cells ; metabolism ; Transgenes ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors ; Virus Assembly

OBJECTIVETo construct the adenoviral vector bringing hVEGF(121) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.

METHODSHuman vascular endothelial growth factor (hVEGF(121)) cDNA obtained from the plasmid pCDI/VEGF(121) was cloned into plasmid pshuttle and further cloned to Adeno-X Viral DNA. The recombinant adenoviral plasmid was identified and then transferred to the adenoviral packaging cell HEK293 by lipofectamine mediated gene transfer method to pack the virus. After titilating the virus, the mouse bone marrow stromal cells (MSC) were transfected by the adenovirus and the expression of VEGF gene was detected.

RESULTSThe recombinant Adeno-VEGF(121) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. After MSCs were tranfected by the virus, RT-PCR showed that hVEGF(121) mRNA was transcripted from the hVEGF(121) gene. Western blot and immune histochemistry showed VEGF(121) protein was expressed in transgene MSCs.

CONCLUSIONThe recombinant adenoviral vector bringing hVEGF(121) cDNA was successfully constructed and the transgene MSC expressed hVEGF gene in vitro, it provided the further foundation of VEGF gene therapy for bone ischemic diseases.

Adenoviridae ; genetics ; Blotting, Western ; Cells, Cultured ; DNA, Complementary ; genetics ; Endothelial Growth Factors ; genetics ; metabolism ; Gene Expression ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Humans ; Immunohistochemistry ; Lymphokines ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors