调节软骨细胞骨架蛋白对成软骨表型的影响

Effects of cytoskeleton modulation on phenotypes of rabbit chondrocytes

目的研究软骨细胞骨架蛋白结构改变与成软骨细胞表型改变的关系。方法采用三维微团培养法培养新西兰兔膝关节软骨细胞,将软骨细胞分为对照组、秋水仙碱组、细胞松弛素B组及丙烯酰胺组,通过秋水仙碱、细胞松弛素B及丙烯酰胺分别调节软骨细胞骨架的微管、肌动蛋白微丝及波形蛋白中间纤丝,从大体形态、组织切片染色、免疫组化染色及RT-PCR分析成软骨细胞表型的改变。结果对照组、秋水仙碱组及细胞松弛素B组细胞较密集、均一,丙烯酰胺组细胞较少、分散。对照组、秋水仙碱组及细胞松弛素B组COLⅡ、蛋白多糖染色深,COLⅠ染色浅;而丙烯酰胺组COLⅡ、蛋白多糖染色浅,COLⅠ染色较深。RT-PCR数据统计分析显示对照组与秋水仙碱组、细胞松弛素B组相比,COLⅠ(1.21±0.21 vs 1.55±0.06、1.70±0.07)及COLⅡ(1.10±0.12 vs 0.91±0.21、0.90±0.30)表达差异无统计学意义(P>0.05);而对照组COLⅡ表达明显多于丙烯酰胺组(0.26±0.06),COLⅠ表达明显低于丙烯酰胺组(9.63±0.38),差异有统计学意义(P<0.05)。结论丙烯酰胺调节软骨细胞骨架波形蛋白中间纤丝可明显下调COLⅡ、蛋白多糖的表达。

Objective To determine the relationship of structure of cytoskeletal proteins with cell phenotypes in chondrocyte. Methods Chondrocytes isolated from the knee joints of New Zealand rabbits were cultured in 3-D pellet, and then divided into control group, colchicine group, cytochalasin B group and acrylamide group. The cells were exposed to colchicine, acrylamide, or cytochalasin B to disrupt tubulin microtubules, vimentin intermediate filaments, or actin microfilaments, respectively. The chondrocyte phenotypes were determined by gross observation, histological staining, immunohistochemistry, and RT-PCR. Results The chondrocytes were in more dense and uniform distribution in control group, colchicine treated group, and cytochalasin B treated group, while those in acrylamide treated group were in dispersed distribution. The expression levels of type Ⅱ collagen and aggrecan were higher, while that of typeⅠcollagen was lower in the control, colchicine and cytochalasin B groups. But their expression profiles were opposite in acrylamide treated group. RT-PCR analysis showed that there was no significant differences in the expression levels of typeⅠcollagen and type Ⅱ collagen in the control group （1.21±0.21, 1.10±0.12） compared to the colchicine group （1.55±0.0, 0.91±0.21） and cytochalasin group （1.70±0.07, 0.90±0.30, P〉0.05）. But the expression of type II collagen was obviously higher, and that of typeⅠcollagen expression was lower in the control than the acrylamide group （1.10±0.12 vs 0.26±0.06, 1.21±0.21 vs 9.63±0.38, P〈0.05）. Conclusion Acrylamide-induced modulation of vimentin intermediate filaments down-regulates the expression of type II collagen and aggrecan in the chnodrocytes.