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Influence of Adenoviruses-mediated Human Vegf and Ang-1 Expression on the Proliferation and Osteogenic Potential of Rat Bone Mesenchyme Stem Cells

Author: ZhangJinKang
Tutor: LiuJian;MengGuoLin
School: Fourth Military Medical University
Course: Surgery
Keywords: Vascular endothelial growth factor Angiopoietin-1 Adenovirus vector Gene transfection Bone marrow stromal cells Proliferation Osteogenic differentiation
CLC: R329
Type: Master's thesis
Year: 2010
Downloads: 42
Quote: 0
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Large bone defects caused by high-energy trauma, osteomyelitis, bone tumors are troubled by the problem of the orthopedic surgeon. Bone tissue engineering provides a good way to study for the treatment of bone defects. Engineered artificial bone implanted in the body the ability to survive depends on the ability to achieve effective vascularization get enough nutrients from the host. The vascularization issues become the bottleneck restricting engineered artificial bone used clinically. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is a specific vascular growth factor, can increase the permeability of the blood vessels, stimulate the proliferation and migration of endothelial cells to promote the formation of new blood vessels. However, the leakage of VEGF-induced neovascularization, immature, difficult to play normal function. The angiopoietin (Angiopoietin-1, ANG-1) play an important role in the shaping of vascular pericytes and smooth muscle cells raised, neovascular structures remain intact. Our group intends to build a two-gene encoding human VEGF165 and ANG-1 adenovirus vector pAd-VIA to promote rapid vascularization of tissue engineered bone implanted after transfection of rat bone marrow stromal cells (BMSCs). However, the double gene transfection, whether the BMSCs biological activity is affected, is unclear. The purpose of this experiment is to study the adenovirus vector pAd-VIA rat BMSCs after transfection, the expression of exogenous gene in vitro situation, and the impact on proliferation and osteogenic differentiation of rat BMSCs transfection, built for the next step has vascular features tissue engineering bone repair large bone defects lay the theoretical foundation. Preparation and titer determination of the weight combination adenovirus vector pAd-VIA purpose of preparation of carrying human vascular endothelial cell growth factor (VEGF165) and angiopoietin -1 (ANG-1) double gene co-expression of the recombinant adenovirus vector pAd-VIA for further experiments to provide the tools for gene transfection. This room constructed encoding human VEGF165 and ANG-1 double gene adenovirus vector P-VIA digested with PacI so linearized turn to use AdEasyTM adenovirus system restructuring and packaging adenovirus green fluorescent protein (GFP) expression of the light microscope, cell morphology evaluation, and a large number of amplification using 50% tissue culture infectious dose (TCID50) method detects virus titer. Adenovirus plasmid transfected QBI-293A cells 8d light microscope visible cells round shrink gathering was like the typical cell colonies pathological change (CPE); visible GFP expression under a fluorescence microscope, and the fluorescence intensity with incubation time and gradually enhanced; obtain high titers of adenovirus vectors through multiple rounds of infection, after amplification. The virus titer of 2 × 1010PFU/ml. Conclusion We successfully constructed recombinant adenovirus vector carrying human VEGF165 and ANG-1 double gene co-expression of pAd-VIA, and lay the foundation for artificial bone vascularization of tissue engineering research. Identification of two pairs of gene transfection in vitro expression of rat BMSCs and explore its proliferation purposes carrying human vascular endothelial cell growth factor 165 (VEGF165) and angiopoietin -1 (ANG-1) dual-gene adenovirus expression vector pAd -VIA transfection in vitro expression of rat bone marrow stromal cells (BMSCs) and BMSCs proliferation. The chamber constructed encoding human VEGF165 and ANG-1 double gene recombination adenovirus vector pAd-VIA vitro transfection rats of BMSCs, through the expression of green fluorescent protein (GFP), Western-blotting, enzyme-linked immunosorbent assay ( ELISA) was used to detect the expression of exogenous gene, multiplicity of infection (multiplicity of infection, MOI), respectively, for 50, 100, 200, 400 with different concentrations of adenovirus transfected BMSCs proliferation activity by MTT assay. Results recombinant adenovirus vector pAd-VIA transfection in vitro rat BMSCs of GFP expression, Western-blotting analysis showed that transfection group VEGF165 and ANG-1 antibody blot bands about 45KD and 14.4KD ; ELISA results show: transfection group 1, 2, 3 d VEGF165 concentration in the supernatant and the concentration of ANG-1 continues to increase, untransfected almost exogenous gene expression was not detected, the difference was statistically significant (P lt; 0.01); different concentrations of adenovirus vector transfected BMSCs promote cell proliferation, cell growth curve shift in 1,3,5,7 d the transfection group of OD value greater than untransfected group, the difference statistically significant (P lt; 0.01), 9d, cells enter plateau transfected and untransfected group OD value difference was not statistically significance (P gt; 0.05). Conclusion The recombinant adenoviral the pAd-VIA transfection rat BMSCs after exogenous gene in vitro has been effectively expressed, at the same time can promote the proliferation of rat BMSCs in the observation time. 3 pairs of gene transfection the rat BMSCs osteogenic differentiation potential purpose research co-transfection of human vascular endothelial growth factor 165 (VEGF165) and angiotensin -1 (ANG-1) double gene of rat bone marrow stromal cells ( BMSCs) osteogenic differentiation potential impact. The room built encoding human VEGF165 and ANG-1 double gene adenovirus vector pAd-VIA vitro transfection rats of BMSCs application of vitamin C, dexamethasone, β-glycerophosphate inducing culture medium oriented induction into bone cell differentiation. The experiment was divided into four groups: A: adenovirus vector pAd-VIA the transfected BMSCs and induced group; B: BMSCs induced group; C: adenovirus vector pAd-VIA transfected BMSCs group; D: pure BMSCs group. Staining and activity was measured by alkaline phosphatase (ALP), collagen type I and osteocalcin immunofluorescence staining, alizarin red staining to evaluate joint transfected gene into rat BMSCs osteogenic differentiation potential impact. Recombinant adenovirus vector pAd-VIA vitro transfection of rat BMSCs group A and group B ALP staining, immunofluorescence staining of collagen type I and osteocalcin, Alizarin red staining was positive, while Group C and Group D negative. Expression of ALP activity, 3d begin to express 7d reach the peak, A, group B and group C and group D ALP expression between 7 and 14d have a statistically significant difference (P lt; 0.01), group A and between Group B, at each point in time, there was no significant difference (P gt; 0.05). Conclusion adenovirus vector pAd-VIA the transfected rat BMSCs not significantly affect the in vitro osteogenic differentiation potential.

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