The Study About The Role And Mechanism Of GDF5 On Chondrocyte Dedifferentiation

Objective:To observe the dedifferentiation phenomenon in cultured articular chondrocytes of C57 mice. To achieve re-differentiation of dedifferentiated chondrocytes after treatment with GDF-5. Take a preliminary study on the MiRNA-145 mechanism in differentiation of chondrocytes. Methods:Primary chondrocytes isolated from the knee-joints of neonatal C57 mice were subcultured to the seventh generation.Commonly used experimental approaches such as cell microscopy, CCK-8 proliferation assay, Alcian blue staining, quantitative real-time PCR and western blot were performed to identify the cell morphology, proliferation, matrix secretion and protein expressionof the cells of generations Pi,P4 and GDF5 treated P4. Meanwhile, Different experiment groups of rabbit chondrocytes combined with PLGA/CHI complex for 7 days were repaired of rabbit knee joint defect. Three months later, articular cartilages were isolated and subjected to HE staining, Alcian blue staining and immunohistochemical staining. The MiRNA- 145 mimic and inhibitor was transfected into chondrocytes, then detect the protein expression level of SOX9 in samples. Results:During the process of subculture, the articular chondrocytes of C57 mice and rabbit were converted; results showed that the proliferation of P4 generation was slightly weakened compared to Pi generation and P7 generation showed no obvious proliferation; Alcian blue staining revealed that the decrease in extracellular matrix secretion was reversed in cartilage cells of P 4 generation treated with GDF5 for 7 days; importantly, consistent results were obtained by qRT-PCR and western blot. It was demonstrated that the PLGA/CHI support bare low cytotoxicity by vital cell staining. Hoechst33258 staining showed that the cells grew into the interior of the microspheres gradually from the surface with the extension of incubation time. In vivo, histology staining was also found that the P4cells +PLGA/CHI support complex group was less repaired than that of generation P1 cells +PLGA/CHI support complex group, while the repair effect was increased in the group filling with generation P4 cells+PLGA/CHI support complex treated with GDF5 compared to the group repaired with generation P4 cells+PLGA/CHI support complex alone. MiRNA-145 regulate the expression of SOX9 during the dedifferentiation of articular chondrocytes, inhibition of MiRNA-145 can enhance the expression of SOX9, equally, over-expression of MiRNA-145 can be reduced SOX9. Conclusions: Obvious dedifferentiation phenomenon appears incultured chondrocytes of generation P4, while, after being treated with GDF-5 for 7 days at a concentration of 300ng/ml, the differentiation phenotype of cultured chondrocytes can be restored partly. Consistent results are obtained in vivo. MiRNA-145 plays a negative role in the regulation of cartilage cells to express SOX9 in the dedifferentiation process.