Effects of Cyclin D1 gene silencing on apoptosis and proliferation of chondrocytes in osteoarthritis:an in vitro study in rats
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摘要: 目的 研究周期蛋白D1(Cyclin D1)表达沉默对骨关节炎(OA)软骨细胞的增殖和凋亡的影响。方法 从健康的成年大白鼠分离软骨细胞,并进行人工培养。采用甲苯胺蓝染和Ⅱ型胶原免疫组化筛选软骨细胞。构建表达Cyclin D1-siRNA的质粒。在软骨细胞中通过IL-1 β诱导产生OA模型。软骨细胞被分为:空白控制组,IL-1 β诱导组(OA模型组),IL-1 β诱导实验组(OA实验组,Cyclin D1-siRNA转染),IL-1 β诱导的阴性控制组(OA阴性控制组,阴性转染)。每组细胞培养了24 h、48 h、72 h、96 h,且用细胞计数试剂盒量化细胞增殖。用流式细胞术检测细胞周期与凋亡。用qRT-PCR和Western blotting检测细胞中Cyclin D1、mRNA和蛋白质的表达。结果 细胞增殖测定结果表明,相较于培养了24 h的软骨细胞,72 h和96 h的软骨细胞增殖速度更加显著。和空白控制组相比,培养72 h和96 h的OA模型组、OA实验组和OA阴性控制组的细胞增殖速度显著下降。OA模型组细胞增殖在48 h、72 h和96 h显著低于OA实验组、OA阴性控制组。流式细胞术表明,相比较于空白控制组,OA模型组、OA实验组、OA阴性控制组中细胞凋亡率更高。相比较于OA模型组和OA阴性控制组,OA实验组细胞凋亡率增加。相关Cyclin D1 mRNA和蛋白质表达,在OA实验组中,显著低于空白控制组、OA阴性控制组和OA模型组。结论 Cyclin D1基因表达沉默能够抑制OA软骨细胞的增殖和增强OA软骨细胞的凋亡。Abstract: 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.
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Key words:
- Cyclin D1 /
- Gene silencing /
- Osteoarthritis /
- Chondrocytes /
- Cell proliferation /
- Cell cycle /
- Apoptosis
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