Experimental Study Of Human Bone Marrow Mesenchymal Stem Cells Transfected With Vascular Endothelial Growth Factor 165 Gene And Its Application In Tissue-engineered Skin

Severe trauma, extensive burns, tumor removal, plastic surgery wound coverage has been plagued clinicians problems. At present, conventional treatment methods have autologous, allogeneic or xenogeneic skin transplantation,Source of autologous skin is extremely limited, and there are defects in the formation of a new wound; Allogeneic or xenogeneic skin graft resolve the problem of inadequate sources, but because it is difficult to overcome immune rejection, could only be used for temporary closure of wound coverings. Thus to carry out search for wound healing of skin substitutes research, have important clinical significance. In recent years, tissue engineered skin substitutes provide an alternative to skin autografts for treatment of nonhealing wounds that often develop in patients with extensive burns and with vascular insufficiency such as venous stasis or diabetes or in the elderly. In present tissue engineered skin has been a hot spot in repair of wounds. While skin substitutes such as Apligraf promote healing, they usually do not stably engraft and significant numbers of patients receiving bioengineered skin substitutes require additional interventions as a result of graft failure. Various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. So how to stimulating vascularization of tissue - engineered skins grafting becomes a critical question and a urgent problem need to address either.Study shows that transgenic technology can import angiogenesis regulatory factor gene into target cells to express certain regulatory factor protein and contribute to angiogenesis.Bone mesenchymal stem cells ( MSCs ) are isolated from bone marrow mononuclear cells and can be expanded ex vivo. Under appropriate culture conditions, human bone marrow mesenchymal stem cells have the capacity to differentiate into cells such as bone, cartilage, adipocytes, myocytes, and even cardiomyocytes. Bone marrow mesenchymal stem cells also have immunomodulatory and anti-inflammatory effects, but only evoke little immune reactivity. Moreover, bone marrow mesenchymal stem cells are amenable to genetic manipulation, thus, these cells are currently being tested for their potential use in cell and gene therapy . Over the past two decades as in-depth study of biological materials and tissue engineering technology, constant improvement of the skin tissue engineering research and applications for clinical repair of various skin defects providing a new way. Vascular endothelial growth factor (VEGF) is the strongest known angiogenesis-promoting factor, In recent years the application of VEGF165 gene transfection of bone marrow mesenchymal stem cells for myocardial ischemia or limb ischemic disease of experimental and clinical studies have achieved initial success. So far, the research about construction tissue - engineered skins by transfection hVEGF165 gene into human bone marrow mesenchymal stem cells has not been reported. Therefore, the use of gene transfer technology to transfected VEGF gene into into bone marrow mesenchymal stem cells and to construct tissue - engineered skin with VEGF- modified human bone marrow mesenchymal stem cells, and covered the skin wound by constructed tissue - engineered skin, so the bone marrow mesenchymal stem cells could endocrine VEGF protein in the local in short-term, thereby contributing to tissue-engineered skin early neovascularization , it is possible to provide a novel gene therapentic strategy for neovascularization which can possibly apply for clinic usage.This study is intended to transfect hVEGF165 gene into human bone marrow mesenchymal stem cells with liposome, Observe VEGF expression in vitro and detect the effect of transfection on human bone marrow mesenchymal stem cells and to construct tissue - engineered skin with VEGF- modified hMSCs as well as acellular dermal and then graft tissue - engineered skin to full thickness dermal wounds of animals, Observed its role in the reconstruction of skin structure and function and its prognosis, compared vascularization of wound defect, to speed up tissue-engineered blood vessels after transplantation of skin, to explore new avenues to accelerate neovascularization of tissue - engineered skin and improve its success rate of clinical transplantation.Therefore, a series of experiment studies were performed as follows: Part I Isolation, cultivation and biological identification of human bone marrow mesenchymal stem cellsOBJECTIVE:The aim of this study is to research on the method to isolate and purify human bone marrow mesenchymal stem cells and provides basis for the amendment of tissue defects by tissue engineering.METHODS:Human bone marrow mesenchymal stem cells were isolated by combining density gradient centrifugation with plastic adherence . Morphological observations were performed with phase contrast microscope; growth curves of the cells were drawn by cytometry method; cell phenotype and generation cycle were detected by flow cytometry; cell ultrastructures were determined by transmission electron microscope.RESULTS:Higher purity of hBMSCs could be achieved by density gradient centrifugation. The positive expression rates of cell phenotypes were various as followings respectively: CD90,98.48%; CD29,98.74%;CD44,97.41%; CD34,0.36%; CD45,0.64%. The growth curve of human bone marrow mesenchymal stem cells was"S"shaped. The ratio of cells in S+G2+M stage of the5th passages was 15.26%. The ratio of nucleus to cytoplasm was large and some organelle and protein secretion could be seen in cytoplasm.CONCLUSION:Higher purity of hBMSCs can be isolated and cultured by combining density gradient centrifugation with plastic adherence. Part II Experimental Study of the transfection of Vascular endothelial growth factor 165 gene into human bone mesenchymal stem cells in vitroOBJECTIVE:To observe the expression of exogenous gene and protein after human bone marrow mesenchymal stem cells transfected with human vascular endothelial growth factor 165 (VEGF165) gene, and to investigate the feasible of promotion tissue-engineered skin early revascularization.METHODS:Bone marrow mesenchymal stem cells were separated and purified by combining density gradient centrifugation with adhering method. The vector pShuttle-CMV/VEGF165 was transfected into bone marrow mesenchymal stem cells by liposome mediated and the bone marrow mesenchymal stem cells were divided into four groups : plasmid group,the empty plasmid group , liposome group and the control group, The cells in the plasmid group were transfected with pShuttle-CMV/VEGF165 ,while cells in the empty plasmid group were transfected with pShuttle-CMV and cells in the liposome group were transfected with Lipofectamine 2000 and cells in the control group received no special treatment. Human VEGF expression in vitro was assessed by RT-PCR,ELISA and Western Blot. MTT method was used to detect the effect of transfection on bone marrow mesenchymal stem cells . Apoptosis was detected by flow cytometry.RESULTS:The expressions of VEGF165 mRNA in each group by RT-PCR : the plasmid group,empty lasmid group,liposome group and the control group the VEGF165mRNA expression were 0.89±0.03,0. 34±0.04,0.40±0.03,0.30±0.03, Compared with other three groups, the VEGF165mRNA transcripts of bone mesenchymal stem cells in the plasmid group were significantly raised (P <0.01).The expression of VEGF165 protein in each group by ELLISA: The content of VEGF in the plasmid group,empty lasmid group,liposome group and the control were 778.39±35.21 pg/ml,543.55±24.32pg/ml,561.45±28.08pg/ml,571.53±22.73pg/ml, Compared with other three groups, the expression of VEGF165 protein in the plasmid group were significantly raised (P <0.01), and in the plasmid group , VEGF expression level gradually increased to the highest on day 7, gradually decreased later. Western Blot showed that the intensity of the plasmid group band increased(P <0.01). It wasn' t found that VEGF transfection had a significantly inhibitory effect on MSCs by MTT method and flow cytometry .CONCLUSION:Bone marrow mesenchymal stem cells were transfected with hVEGF165 gene successfully and deffectively express target gene and protein , provide a novel gene therapentic strategy for neovascularization which can possibly apply for clinic usage. Part III Construction of tissue - engineered skin with vascular endothelial growth factor - modified bone marrow mesenchymal stem cells and acellular dermal matrixOBJECTIVE: To construct tissue - engineered skin with VEGF- modified hBMSCs as well as acellular dermal matrix.METHODS:Bone marrow mesenchymal stem cells were separated and cultured in vitro.The vector pShuttle-CMV/VEGF165 was transfected into bone marrow mesenchymal stem cells by liposome mediated at the time of 80%- 90% confluence and the VEGF- modified bone marrow mesenchymal stem cells were seeded onto the surface of acellular dermal matrix. It was cultured absolutely inside liquid medium for about 2d. In this way, tissue - engineered skin was expected to be constructed in vitro. Observation the cells seeded to scaffold material and its compatibility.RESULTS:With the VEGF- modified cells cultured in the acellular dermal matrix, tissue - engineered skin could be construted in virto.CONCLUSION: Constuction of tissue - engineered skin with VEGF- modified bone marrow mesenchymal stem cells as well as acellular dermal matrix in vitro is feasible. Part IV Experimental study of the transplanted human bone mesenchymal stem cells transfected with geneaccelerate the neovascularization of tissue - engineered skin OBJECTIVE:To investigate the feasibility of transplanting VEGF- modified tissue - engineered skin for accelerating neovascularization of tissue - engineered skin .METHODS:Twenty-six New Zealand white rabbits with four full thickness skin defects on the two sides of the back were separately covered with hVEGF165- MSCs+ADM, MSCs+ADM , ADM or none. Examine wound healing of local tissue and count it's MVD by CD34 immunocytochemistry staining.RESULTS:1 week after operation the capillary density of wound tissue of VEGF gene transfection treatment group was significantly higher than those of MSCs+ADM treatment group and ADM treatment group ( P < 0.01). 2 weeks after operation the capillary density of wound tissue of VEGF gene transfection treatment group was highest in those three groups, However, among the three groups no significant difference. At the second week and third week after operation, There was significant difference in the survival rate and contraction rate between the three groups, Compared with the two groups, the graft survival rate of VEGF gene transfection treatment group was highest and the contraction rate was lowest ( P < 0.01).CONCLUSION:Transplantation tissue-engineered skin constructed by human bone mesenchymal stem cells transfected with gene VEGF and acellular dermal matrix can promote tissue-engineered skin early revascularization and wound healing effectively.