Influence Of Adenoviruses-mediated Human Vegf And Ang-1 Expression On The Proliferation And Osteogenic Potential Of Rat Bone Mesenchyme Stem Cells

The repair of large bone defect caused by high energy trauma,bone tumor and osteomyelitis is a big problem to the orthopaedic surgeon. Bone tissue engineering is a promising field which can help to solve this problem. According to the previous research, vascularization of the artificial bone plays a significant role in bone fracture healing. Therefore, how to vascularize the artificial bone became the bottle-neck of repairing large bone defect. Vascular endothelial growth factor (VEGF) is one of the special factors associated with the growth of blood vessels. It can increase blood vessels'permeability,stimulate proliferation and migration of endothelial cells and promote formation of new blood vessels. Otherwise, the new blood vessels induced by VEGF was leakage and immature and it can not functioned well. Angiopoietin-1(ANG-1) is important in the remolding of new blood vessles. It can recruit perivascular cells and smooth muscle cells and maintain the integrity of new blood vessles. Ang-1 can offset the leakage of the new blood vessles induced by VEGF. We have constructed the recombinant adenovirus vector pAd-VIA co-expressing human VEGF165 and Ang-1 genes successfully. We expected to combine rat bone mesenchyme stem cells transfected by pAd-VIA and scaffold in vitro and implant them to promote vascularization of the artificial bone in early stage. Whether the proliferation and osteogenic potential of rat bone mesenchyme stem cells will be influenced is unclear. At present, we transfected rat BMSCs with pAd-VIA, and observed the expression of the interest genes in vitro. Meanwhile, we evaluated the proliferation and osteogenic potential of rat BMSCs infected by pAd-VIA.1. Establishment of recombinant adenovirus vectors pAd-VIA containing vascular endothelial growth factor and angiopoietin-1Objective: the purpose of this study was to construct the recombinant adenovirus vector co-expressing human vascular endothelial growth factor 165(VEGF165) and angiogenin-l(Ang-1) in order to providing gene transfer tools for the next experiment. Methods: we have constructed the adenovirus plasmid encoding VEGF165 and ANG-1 in our laboratory. Further the recombinant plasmid was packaged and amplified in QBI-293A cells after Pac I digestion to get adenovirus vector pAd-VIA. The package of the gene of interests was evaluated by fluorescence microscopic analysis of GFP expression and by microscopic observation. Then, the adenovirus vectors pAd-VIA have been amplified and the titer was tested by TCID50 method. Results: After linearization by Pac-I, the recombinant adenovirus plasmid were packaged and amplified successfully in QBI-293A cells. High positive GFP was expressed in the field through fluorescence microscopy after transfected by recombinant pAd-VIA vectors and the cells shape showed classical CPE change in 8 days. The titer was 2×1010 PFU/ml after amplifying for 4 cycles. Conclusions: The recombinant adenovirus vectors containing vascular endothelial growth factor 165 and angiopoietin-1 were successfully constructed, thus providing convenient gene transfer tools for constructing the new gene-modified artificial bone. 2. Effect of adenoviruses-mediated human angiopoietin-1 and VEGF expression on ex vivo growth capability of bone mesenchyme stem cells in ratObjective: The purpose of this study was to identify expression of the interest genes and to investigate the effect of adenoviruses-mediated human angiopoietin-1 and VEGF165 expression on ex vivo growth capability of bone mesenchyme stem cells (BMSCs) in rat. Methods: The recombinant plasmid constructed by our laboratory was packaged and amplified in QBI-293A cells. The recombined adenovirus pAd-VIA was obtained. Rat BMSCs were infected with recombined adenovirus pAd-VIA at about 80%confluency. The expression of gene of interests was evaluated by fluorescence microscopic analysis of GFP expression,western blotting and enzyme 1inked immunosorbent assay(ELISA). The impact of transfection of adenovirus vector pAd-VIA with different concentration (MOI=50,100,200,400) on the proliferation rate of BMSCs was detected by MTT automated colormetric microassay. Results: we packaged and amplified the recombinant adenoviruses vectors pAd-VIA successfully. High positive GFP was expressed in the field of BMSCs through fluorescence microscopy after transfected by recombinant vectors pAd-VIA. Western-blotting and ELISA indicated stable protein expression. The results showed that the concentration of VEGF165 and ANG-1 was increasing gradually in transfected groups and was no increase in non-transfeced groups. The difference was significant (P<0.01). It was indicated by MTT that the growth of BMSCs transfected with adenovirus vectors pAd-VIA was higher than non-transfected cells in 1,3,5,7d (P<0.01), but there were no significant diference in 9d between the transfected and non-transfected groups (P>0.05). Conclusion: The growth capability of rat bone mesenchyme stem cells (BMSCs) infected with adenoviruses vectors encoding human VEGF165 and ANG-1 was improved.3. Effect of adenoviruses-mediated human angiopoietin-1 and VEGF165 expression on ex vivo osteogenic potential of bone mesenchyme stem cells in ratObjective: The purpose of this study was to investigate the effect of adenoviruses-mediated human angiopoietin-1(ANG-1) and VEGF165 expression on ex vivo osteogenic potential of bone mesenchyme stem cells in rat. Methods: The recombinant plasmid constructed by our laboratory was packaged and amplified in QBI-293A cells. The recombined adenovirus pAd-VIA was obtained. Rat BMSCs were infected with adenoviruses encoding VEGF165 and ANG-1 at about 80% confluency. The induced culture medium consisted of vitamin C, dexamethasone andβ-sodium glycerophosphate. The osteogenic potential of transfected and non-transfected cells in osteogenically differentiated or non-differentiated medium were determined by Alkaline phosphate staining,Alkaline phosphate activity assay,collagen I and osteocalcin immunofluorescence staining,alizarin red staining. Results: we packaged and amplified the recombinant pAd-VIA vectors successfully. High positive GFP was expressed in the field through fluorescence microscopy after transfected by recombinant vectors pAd-VIA. The transfected and non-trasfected cells in osteogenically differentiated medium were positive in Alkaline phosphate staining,collagen I and osteocalcin immunofluorescence staining and alizarin red staining. Compared with the cells in non-differentiated medium, ALP activity in transfected or non-trasfected cells in osteogenically differentiated medium was increased significantly (P<0.01). After infected with the recombined adenovirus, there were no significant difference of ALP activity between the cells transfected and not transfected in osteogenically differentiated medium (P >0.05). Conclusions: The pAd-VIA recombined adenovirus vectors were obtained successfully. After being transfected, the osteogenic potential of rat bone mesenchyme stem cells (BMSCs) had not been affected in the observed time.