SHI Zhibin , FAN Lihong , QIANG Hui , TANG Yilun , WANG Kunzheng , DANG Xiaoqian.
  • The First Department of Orthopedics, the Second Affiliated Hospital of Medical College, Xi’an Jiaotong University,Xi’an Shannxi, 710004, P.R.China.;
Recombinant adeno-associated virus; Human vascular endothel ial growth factor 165; Human bone morphogenetic protein 7; Co-expressing; Biological activity
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Objective To study the biological activity of recombinant adeno-associated virus vector (rAAV) coexpressing human vascular endothel ial growth factor165 (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 the14th 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 umbil ical vein endothel ial cells (HUVEC). The biological activity of hBMP-7 was assessed by mineral ization of BMSCs detected by ALP staining and al izarin red staining. Results With infecting time, the hVEGF165 and hBMP-7 expressions increased gradually in two groups, showing significant difference between two groups (P  lt; 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 exprimental 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, prol iferation and tube formation in experimental group. There was significant difference in the number of blood vessel between two groups (P  lt; 0.05). The ALP staining showed more bly stained granules in experimental group than in control group. There was significant difference in the number of the mineral ized nodules between two groups (P  lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has good biological activity in vitro.

Citation： SHI Zhibin,FAN Lihong,QIANG Hui,TANG Yilun,WANG Kunzheng,DANG Xiaoqian.. STUDY ON BIOLOGICAL ACTIVITY OF RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR COEXPRESSINGHUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR 165 AND HUMAN BONE MORPHOGENETIC PROTEIN 7 GENES IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2010, 24(4): 415-419. doi: Copy