Research In Mechanisms Of Growth And Differentiation Factor 5 Promoting Chondrogenic Differentiation Of Mouse Bone Marrow Mesenchymal Stem Cells

PartⅠExpression and Significance of GDF-5 During Limb Skeletal Development of MiceObjective To investigate the expression trend and effect of growth differentiation factor 5 (GDF-5) during limb skeletal development of mice.Methods Mouse embryos were obtained from mice of Kunming strains provided by Department of Experimental Animal of Tongji Medical College of Huazhong Science and Technology University. The day a vaginal plug was found was considered as embryonic day 0.5 (E0.5). During E11.5-15.5 embryos were removed from the pregnant female mice and limb buds were taken out of them to be kept in reserve for use with ophthalmic scissors.The expression of GDF-5 mRNA and protein of mouse fetal limb buds were detected in embryonic days 11.5-15.5(E11.5-15.5) by RT-PCR and Western blotting respectively.Results①Expression of GDF-5 mRNA in mouse embryonic limb buds: A 219bp of specific electrophoretic strip was observed in every limb bud and average light density ratio of GDF5/β-actin was 0.521±0.023, 1.210±0.014, 1.221±0.008, 0.556±0.014, 0.510±0.019 respectively from E11.5 to E15.5. The results demonstrate that the expression of GDF-5 mRNA was constant during early stage of developmental skeletogenesis and began with embryos E11.5, highlighted at embryos E12.5 and E13.5, subsequently dropped at embryos E14.5 and E15.5. There was very significant difference (p<0.01) in average light density ratio of GDF-5/β-actin between E12.5-13.5 and the other three days.②Expression of GDF-5 protein in mouse embryonic limb buds: The expression of GDF-5 protein has a similar change trend as contrast to mRNA from E11.5 to E15.5 and a 13.7KDa protein stripe was detected in every sample. Average light density ratio of GDF5/β-actin protein was 0.589±0.021, 1.103±0.124, 1.112±0.041, 0.601±0.015, 0.552±0.006 respectively from E11.5 to E15.5. Here, we found very significant difference (p<0.01) between in average light density ratio of GDF-5/β-actin protein between E12.5-13.5 and the other three days.Conclusion GDF-5 could enhance chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro, which played an important role in limb skeletal development and joint formation.PartⅡConstruction of Mammal Expression Plasmid pcDNA3.1(+)/GDF -5 and Its Expression In Bone Marrow Mesenchymal Stem Cells of MiceObjective To construct mammal expression plasmid pcDNA3.1(+)/GDF-5 and check the expression of it in bone marrow mesenchyal stem cells of mice.Methods Two primers were chemosynthesized according to the mouse GDF-5 sequence reported in Genbank. The GDF-5 gene was obtained by RT-PCR from cDNA which was isolated from mouse embryonic limb buds at embryos 14.5, and merged into the pcDNA3.1(+) vector. Then the recombined plasmid was transformed into E.coli host strain DH5α. The double-stranded DNA of the positive clone was analyzed by restriction endonuclease mapping and DNA sequencing. Then the recombined plasmid was transfected into mouse bone marrow mesenchyal stem cells. Expressions of GDF-5 gene and protein were detected by RT-PCR and immunocytochemistry. Results Digestion of the recombinant plasmid with double restriction enzyme showed about two specific electrophoretic strip with 1.6kbp and 5.4kbp. The sequence of mouse GDF-5 was consistent with that reported in Genbank. With cells transfected by the expression plasmid pcDNA3.1(+)/GDF-5, expressions of the GDF-5 gene and protein were detected positively, but the untransfected cells were negative. Conclusion The recombinant mammal expression plasmid, pcDNA3.1(+)/GDF-5 can be successfully constructed and expressed in bone marrow mesenchyal cells. This provides a basis for further research on GDF-5 gene in the mechanism of chondrogenesis or cartilage and bone tissue engineering.PartⅢGrowth Differentiation Factor 5 Enhances Chondrogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells In VitroObjective To explore the effect of Growth Differentiation Factor 5 on chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro. Methods The MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were chosen to induce into chondrogenic differentiation with different concentration of recombinant human GDF-5(0ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml). After 14 days of induction, morphology of cells was observed under phase-contrast microscopy. The proliferation effect of MSCs was investigated by MTT assay. TypeⅡcollagen mRNA and protein were examined with RT-PCR and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue.Results According to MTT assay GDF-5 had no effect on the proliferation of MSCs; RT-PCR showed that GDF-5 could promote expression of TypeⅡcollagen mRNA in dose dependant manner, especially strong with the concentration of 50ng/ml and 100ng/ml, and immunocytochemistry exhibited that the protein had a similar change. Histological staining proteoglycan of Alcian blue revealed deposition of typical cartilage extracellular matrix. Conclusion These results suggests mouse bone marrow mesencymal stem cells can be differentiated into chondrogenic phonotype with the induction of GDF-5 in vitro, and which provides a basis for further research on the mechanism of GDF-5 in chondrogenesis.PartⅣEffect of Growth Differentiation Factor 5 on Connexin43 Expression With Chondrogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells In VitroObjective: To investigate the effect of Growth Differentiation Factor 5 on gap junctional protein connexin43 expression with chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro.Methods: The MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were chosen to induce into chondrogenic differentiation. After 72 hours of induction, TypeⅡcollagen protein was examined with immunocytochemistry and the sulfate glycosaminoglycan was measured by Alcian blue. With 24,48 and 72 hours of induction, the proliferation effect of MSCs was investigated by MTT assay; Connexin43 mRNA and protein were examined with RT-PCR, western blotting and immunocytochemistry respectively in different time points of induction.Results: According to MTT assay GDF-5 had no effect on the proliferation of MSCs in different time points of induction; RT-PCR, western blotting and immunocytochemistry showed that GDF-5 could promote expressions of connexin43 mRNA and protein in different time of induction, especially highlighting after 48 hours of induction. After 72 hours of induction, immuncytochemistry showed expression of TypeⅡcollagen protein, and histological staining proteoglycan of Alcian blue revealed deposition of typical cartilage extracellular matrix.Conclusion: These results suggest GDF-5 can enhances chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro through up-regulating the expression of gap junctional protein connexin43.PartⅤEffect of gap junctional blocker 1-heptanol on chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitroObjective To investigate the effect of gap junctional blocker 1-heptanol on chondrogenic differentiation of bone marrow mesenchymal stem cells with the induction of GDF-5 in vitro.Methods The MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were chosen to induce into chondrogenic differentiation with recombinant human GDF-5(100ng/ml) or 1-heptanol(2.5μmol/L). The proliferation effect of 1-heptanol on MSCs was investigated by MTT assay. TypeⅡcollagen mRNA and protein were examined with RT-PCR and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue. Connexin43 protein was examined with western blotting.Results MTT assay showed that 1-heptanol had no effect on the proliferation of MSCs; RT-PCR and immunocytochemistry all showed that 1-heptanol could inhibit typeⅡcollagen mRNA and protein alternatively expressed by MSCs with the intervene of GDF-5 and 1-heptanol. Alcian blue staining revealed that 1-heptanol could inhibit deposition of typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol play no effect on the expression of connexin43.Conclusion These results suggest that mouse bone marrow mesencymal stem cells can be differentiated into chondrogenic phonotype with the induction of GDF-5 in vitro, and that connexin43-conaining gap junction cellular communication plays an important role in chondrogenesis with the induction of GDF-5.