Résumé
Objective To investigate the effect of growth differentiation factor 5(GDF-5)on expression of gap junctional protein,connexin 43,during ehondrogenic differentiation of bone marrow sternal cells(BMSCs)in vitro.Methods BMSCs were isolated from mouse bone marrow and cultured in vitro.The cells in passage 3 were chosen to be induced into chondrogenic differentiation.After induction for 72 hours,TypeⅡcollagen protein was examined by immunocytochemistry and the sulfate glycosaminoglycan was measured by Alcian blue staining.With induction for 24,48 and 72 hours,the proliferation effects of BMSCs were investigated by MTT assay;connexin 43 mRNA and protein were examined by RT-PCR,western blotting and immunocytochemistry respectively at different time points during induction.Results According to MTT assay,GDF-5 had no effect on the proliferation of BMSCs at different time points of induction;RT-PCR,western blotting and immunocytochemistry showed that GDF-5 could promote expressions of connexin 43 mRNA and protein at different times during induction.After 72 hours of induction,immunocytochemistry showed expression of TypeⅡcollagen protein,and AIcian blue staining of proteo- glycan revealed deposition of typical cartilage extracellular matrix.Conclusion GDF-5 can enhance chondrogenic differentiation of mouse BMSCs in vitro by up-regulating the expression of gap junctional protein,connexin 43.
Résumé
Objective To investigate the effects of gene transfection with human growth/differentiation factor 5(hGDF5)on the differentiation and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs).Methods BMSCs were obtained from adult New zealand rabbits and purified by gradient centrifuge.Exogenous recombinant human GDF5 was transfected into BMSCs with liposome method.Then hGDF5 expression at mRNA and protein level was measured separately by reverse transcription-polymerase reaction and indirect immunofluorescence method.Activity of alkaline phosphates(ALP),expression of TypeⅡcollagen(ColⅡ),proteoglycan and growth of the cells were all measured by biological methods to evaluate the effects of hGDF5 gene transfer on the differentiation and proliferation of rabbit BMSCs.Results After hGDF5 gene transfection,BMSCs expressed hGDF5 mRNA and protein,and compared with the control groups,expression of proteoglycan and ColⅡ increased significantly,but no significant difference appeared in ALP activity and cell proliferation.Conclusion Gene transfer with hGDF5 is an effective way to enhance the expression of GDF5 at mRNA and protein.The expression of heterogenetic hGDF5 gene can induce BMSCs' differentiation to chondrogenic cells.But the gene transfection has no obvious effects on the proliferation and ALP activity of BMSCs.