Effect Of TWEAK Mediated By Adenovirus Vector On Rats Bone Mesenchymal Stem Cells

Much bone tissue defects due to trauma, tumor and other factors will cause not only a partial defect of the deformity, but also the loss of function, which eventually affect patients'life quality. The routine autology and allograft bone transplantation have no satisfactory effectiv- eness. With the development of molecular biology technique ,vascularize tissue engineered bone is the solution to the problem. Vascularize artificial bone needs special seed cells. Bone marrow-derived mesenchymal stem cells has multi-differentiational potentialities, it is easy to form a directional differentiation under definited inducing conditions and can be infected with the foreign gene easily to secrete ,so it is the most important seed cell in the construction of vascularized tissue engineering bone. The proliferation and directional differentiation of seed cells are always the difficult and focus problem in the construction of vascularized tissue engineering bone. The key problem is how to promote the proliferation ,directional differentiation and to construct the functional tissue. The successful vascularized tissue engineering bone need two essential factors: the osteogenic differentiation of the seed cells and angiogenesis. The previous studies focus on loading stem cell and vector to the bone defects, and using one or more drugs to stimulate the bone regeneration, Recently, the researchers'attention is placed on using the gene which carried by seed cells to form a paracrine of seed cells for their proliferation and differentiation induction. Tumor necrosis factor–like weak inducer of apoptosis (TWEAK) is the member of the TNF superfamily. The in vitro experiments have shown that TWEAK can promote the proliferation and immigration of the epithelial cells,promote the activity of FGF-2and VEGF-A,and play an important role on the growth and remodeling. From this it is suggested that TWEAK signaling play an important role on the regeneration of the vascular after the damage of bone tissue.The purpose of this experimental study is to transfected the BMSCs with the adenovirus vector taking the TWEAK gene ,to examine the effect of the TWEAK on the proliferation and osteogenic and epithelial differentiation of the BMSCs.The following is the methods:First, The BMSCs was isolated from the femoral bone of rats,cultured and purified to obtain enough cells. Second, The BMSCs was transfected with the adenovirus vector carrying the EGFP and the transfection efficiency was detected. At the same time, BMSCs was transfected with the Adenovirus vector carrying and the TWEAK mRNA was examined by RT-PCR,The effect of the TWEAK on the proliferation using MTT and cell counting.Fourth, Alizarin Red staining was carried out. The content of the alkaline phosphates protein was detected using the Alkaline Phosphates'kit , the I–collage secretion was detected to make sure the different of BMSCs before and after transfection byRT-PCR . Fifth,the CD34 andⅧfactor was detected with the immunohistochemical staining before and after transfection. The BMSCs were cultured on the collagen to determine the growth of MSCs to make sure that if the TWEAK had effect on the BMSCs's endothelial differentiation.Experiment results showed that bone marrow adherent method can success to obtain BMSCs. When the Ad5CMV-EGFP in 300 particles/cell,the transfection efficiency can reach 85%,and when the mass of three generations can also detect 30% fluorescent cells. RT-P showed that TWEAK can transfected into BMSCs,and has high expression. MTT assay and cell counting showed that TWEAK can stimulate the MSCs proliferation, the different between transfected Ad5CMV-EGFP group or untransfected group has Statistical significance(P<0.05). Alizarin red-chip and alkaline phosphatase secretion staining showed that after transfection Ad5CMV-TWEAK ,the experimental group and control group has not statistically significance (P>0.05). RT-PCR which detected the collagen I secretion showed that there was no significant difference (P> 0.05) between experimental group and control group. All of these have proved that TWEAK has no effect on BMSCs osteoblast differentiation. But TWEAK gene transfected BMSCs can induce endothelial differentiation and experimental observation of the growth of transfected cells on the college can see lumen-like structure, indicating that cells transfected TWEAK can promote angiogenesis.The innovation of this study is that Ad5CMV-TWEAK can infect the BMSCs with high efficence, TWEAK gene could not induce the BMSCs into osteoblast, but do induce BMSCs into endotheliocyte and promote the formation of vasoformation. The results gives the theoretical and experimental foundation and would become a new strategy to restructure of bone tissue defects.