不同浓度的Chir99021对软骨细胞增殖和Ⅱ型胶原表达的影响

Effect of Different Concentrations of Chir99021 on Proliferation and Col2a1 Expression of Neonatal Rat Chondrocytes

目的:探讨不同浓度的Wnt/β-catenin信号通路激活剂Chir99021对体外培养的大鼠软骨细胞增殖和Ⅱ型胶原表达的影响。方法:新生24hSD大鼠8只(雌雄不限),取出关节软骨进行软骨细胞培养,细胞培养48h后进行软骨细胞免疫荧光鉴定。以不同药物浓度将软骨细胞分为四组,分别加入浓度为0.00μmol/L(对照组),0.75μmol/L,1.50μmol/L,3.00μmol/L的Wnt/β-catenin信号通路激活剂Chir99021进行干预。24h后分别观察不同浓度组细胞形态的变化,蛋白印迹分析法(Western blot)检测Ⅱ型胶原、β-catenin和PCNA蛋白的表达情况。结果:在0.00μmol/L(对照组),0.75μmol/L,1.50μmol/L,3.00μmol/L的Wnt/β-catenin信号通路激活剂Chir99021干预下,各组细胞数量明显逐渐增加,与对照组相比较,各加药组β-catenin和PCNA蛋白的表达逐渐上调(P<0.05),而Ⅱ型胶原蛋白的表达逐渐下降(P<0.05)。结论:不同浓度的Chir99021激活Wnt/β-catenin信号通路后明显促进了软骨细胞的增殖,却抑制了软骨细胞分泌的Ⅱ型胶原。

Objective：To investigate the effect of different concentrations of chir99021 on the proliferation and the expression of typeⅡcollagen（Col2a1）of rat chondrocytes.Methods：Eight 24 hold SD rats（female or male）were executed by dislocating.The articular cartilage of rats were removed and cuted into blocks of 1mm×1mm size.The articular cartilage were digested by type Ⅱcollagenase for four minutes five times.The mixed cells were cultured in thermostat incubator with 5%CO2 under the condition of 37 ℃.Chondrocytes were identificatied by the immunofluorescence.Rat chondrocytes were cultured with different concentrations of chir99021 for 24h.The concentrations were 0.00μmol/L,0.75μmol/L,1.50μmol/L,3.00μmol/L.Morphological changes,cell proliferation and protein expression levels of PCNA,β-catenin and Col2a1 were investigated and compared among four groups.Results：The chir99021 obviously promoted the proliferation of chondrocytes of newly born（within 24h）SD rats and the promotion rate was associated with the concentration of chir99021.Compared with control group,the expression levels of both PCNA andβ-catenin were upregulated while the expression level of Col2a1 was downregulated with the increasing concentration of chir99021（P〈0.05）.Conclusion：The different concentrations of chir99021 obviously promoted the proliferation of chondrocytes and inhibited the expression of typeⅡcollagen through activating Wnt/β-catenin signaling.