Objective To investigate the effects of Cyclin D1 silencing on proliferation and apoptosis of chondrocytes in osteoarthritis(OA).
Methods Chondrocytes were separated from healthy SD rats and cultured. Toluidine blue staining and type Ⅱ collagen immunohistochemical staining were used to identify chondrocytes. Cyclin D1 -siRNA expression plas- mid was constructed and OA model was induced byIL-1 βin chondrocytes. Chondrocytes were divided into the blank group, IL-1 β-induced group(OA model group), IL-1 β-induced experimental group(OA trial group, transfected withCyclin D1 - siRNA), IL-1 β-induced negative control group(OA negative control group, transfected with negative control sequence). Cells in each group were incubated for 24 h,48 h,72 h and 96 h and cell proliferation was measured by CCK-8. Flow cy- tometry was used to detect cell cycle and apoptosis. qRT-PCR and Western blotting were used to detect Cyclin D1 mRNA and protein expressions in cells.
Results Cell proliferation assay results showed that the proliferation after chondrocytes were incubated for 72 h and 96 h increased significantly in each group compared to those incubated for 24 h. When com- pared with the blank group, cell proliferations in OA model, OA trial and OA negative control groups decreased distinctive- ly at 72 h and 96 h. The cell proliferation at 48 h,72 h and 96 h were significantly lower in the OA model group than in the OA trial and OA negative control groups. Flow cytometry showed that compared with the blank group, higher apoptosis rate was found in the OA model, OA trial and OA negative control groups. In comparison to the OA model group and OA negative control group, the OA trial group had increased apoptosis rate. Relative Cyclin D1 mRNA and protein expressions in OA experimental group was significantly lower than the blank and OA negative control and OA model groups.
Conclusion Cyclin D1 gene silencing can suppress proliferation and enhance apoptosis of chondrocytes in OA.
DownLoad: