[Study on biological activity of recombinant adeno-associated virus vector co-expressing human vascular endothelial growth factor 165 and human bone morphogenetic protein 7 genes in vitro].
OBJECTIVE
To study the biological activity of recombinant adeno-associated virus vector (rAAV) co-expressing human vascular endothelial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes in vitro so as to provide a new method for the therapeutics of osteonecrosis.


METHODS
The 3rd passage rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group). The expressions of hVEGF165 and hBMP-7 were detected by ELISA assay at the 1st, 2nd, 3rd, 7th, 14th days and Western blot assay at the 14th day after transfection. The expression consistencies of hVEGF165 and hBMP-7 were observed by immunofluorescence assay at the 14th day after transfection. The biological activity of hVEGF165 was assessed by angiopoiesis experiment of the 3rd passage human umbilical vein endothelial cells (HUVEC). The biological activity of hBMP-7 was assessed by mineralization of BMSCs detected by ALP staining and alizarin red staining.


RESULTS
With infecting time, the hVEGF165 and hBMP-7 expressions increased gradually in two groups, showing significant difference between two groups (P < 0.05). The expressions of hVEGF165 and hBMP-7 were positive in experimental group and negative in control group, respectively. Immunofluorescence assay showed positive expressions of hVEGF165 and hBMP-7 in the experimental group and negative expression in the control group, the expression of hVEGF165 and hBMP-7 had good consistencies. hVEGF165 secreted from BMSCs enhanced HUVEC migration, proliferation and tube formation in experimental group. There was significant difference in the number of blood vessel between two groups (P < 0.05). The ALP staining showed more strongly stained granules in experimental group than in control group. There was significant difference in the number of the mineralized nodules between two groups (P < 0.05).


CONCLUSIONS
The rAAV-hVEGF165-IRES-hBMP-7 has good biological activity in vitro.
Shi
Zhibin
Z
The First Department of Orthopedics, the Second Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an Shannxi, 710004, P.R. China. jackky9999@sohu.com
Fan
Lihong
L
Qiang
Hui
H
Tang
Yilun
Y
Wang
Kunzheng
K
Dang
Xiaoqian
X
D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D055419:Bone Morphogenetic Protein 7 / Q000096:biosynthesis* / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D011817:Rabbits; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A