Elf-Induction Of Rabbit Marrow Stromalstem Cells Into Chondrocytes By Transfection With Reconstructed Gene In Vitro

Objective Examine transient and stable transfecting the marrow stromal stem cells (MSCs) with the reconstructed pcDN A3-transforming growth factor-β1(TGF-β1)gene TGF-β1gene expression and to evaluate the feasibility of MSCs to the chondrocytes in vitro so as to provide a scientific and experimental basis for afurther "gene enhanced tissue engineering" research.Methods By Percoll density gradient centri-fuge andmonolaryer culture in vitro MSCs were isolated from rabbit bone marrow and were purified. MSCs quality was evaluated with immunohitotochemistry. The cultured 3 rd generation rabbit marrow stromal stem cells(MSCs) was transfected with the reconstructed pcDNA3-TGF-β1 gene by the Liposomes Method, By the immunoh istochemistry method (SABC) to examine the transient transfecting MSCs TGF-β1 gene expression. Positive clones were selected by G418; By the immunoh istochemistry method (SABC) to examine the stable TGF-β1 gene expression. Continous cultured the cells and observed the cells through inerted microscope constantly and took records. By the immunohistochemistry method (SABC), the antigens collagenⅡwere examined at 2 and 4 weeks of the cell culture after transfection with PGL3-TGF-β1gene.The pictures of the immunohistochemistry slice were analyzed with the analysis instrument, and the statistical analysiswas performed with the software of the SPSS 11.0, compared with the control group and the blank group.Results The model of culture and expansion of MSCs in vitro were established.Transfection of the cultured rabbit MSCs in vitro with the reconstructedpcDNA3-TGF-β1 gene by the Liposomes Method achieved a success, By the immunohistochemistry method (SABC) to study TGFβ1, positive particles were detected in the experimental group, but there were no positive particles in the control and the blank groups. There was a significant difference between the two groups of the experiment and the control group based on the analysis of the t-test (P< 0.01) Positive clones were selected by G418. study TGFβ1 positive particles were still detected in the stable experimental group.2 weeks later,by the immunohistochemistry method (SABC) to study collagenⅡ, there were more positive particles in the transfected cells in the experimental group than in the control and the blank groups,4 weeks later, the cells shape became trilateral. the immunohistochemistry method (SABC) to study collagenⅡshowed the different more clearly. There was a significant difference between the experimental group and the two other groups based on the t-test (P<0.05).Conclusion Transfection of the rabbit MSCs with the reconstructed pcDNA3-TGF-β1 gene by the Liposomes Method is successful. The transfected BMS cells with pcDNA3-TGF-β1 gene can express and excrete TGF-β1 when cultured in vitro. The transfected MSCs that secret TGF-β1 can be self-induced into the chondrocytes after being infected when cultured in vitro.