猪软骨细胞老化过程中Aggrecan的表达及其糖链分子结构的改变

Ageing changes in the expression and molecular structure of Aggrecan in cultured chondrocytes

本实验旨在探讨体外培养软骨细胞在老化过程中 ,aggrecan的表达水平与分子结构的变化及其间的相互关系。取体外培养P1～P5各代猪耳软骨细胞 ,RT PCR检测aggrecan球间区域 (interglobulardomain ,IGD)mRNA的表达状况 ;阿利新兰 (AlcianBlue)法检测糖胺聚糖 (glycosaminoglycan ,GAG)的含量和糖链的分子结构。结果显示aggrecanIGDmRNA的表达在P4、P5细胞内明显降低 (P <0 0 5 ) ;GAG的合成也在P4、P5时显著减少 (P <0 0 5 ) ,但这 2项指标的变化趋势之间无显著相关性。而长链 高度硫酸化的GAG由P4软骨细胞开始 ,绝对含量和相对含量均显著减少 (P <0 0 1) ,其与aggrecanIGDmRNA的变化趋势有显著相关性 (P <0 0 1)。由此可见 ,体外培养软骨细胞中ag grecan的表达合成和细胞老化有密切关系。aggrecanIGDmRNA的表达降低与aggrecan中长链 高度硫酸化GAG的合成代谢过程受阻可能是影响老化细胞成软骨能力的重要因素之一。

To investigate the changes in the expression and molecular structure of aggrecan in aging process of cultured chondrocytes. Confluent hog chondrocytes from different passages (P1～P5) were harvested. The expression of mRNA encoding the aggrecan interglobular domain (IGD) peptide was measured by RT PCR. Alcian Blue precipitation was utilized for quantification and structural analysis of glycosaminoglycans(GAGs). The absorbance of complexes was assayed at 600 nm. Results revealed that the mRNA expression level of the IGD was lower ( P <0.05) followed by a decreased synthesis of GAGs ( P <0.05) in P4 and P5 chondrocytes. But there was no significant correlation between them. The amount of longer chain/higher sulfated GAGs as well as their percentage in total GAGs significantly reduced in P4 and P5 chondrocytes ( P <0.01), that was correlated with the change of IGD ( P <0.05). It was demonstrated that the decreased expression of aggrecan was closely associated with cell aging in the chondrocytes in vitro . The reduction of IGD mRNA and the reduction of aggrecan with longer chain/higher sulfated GAGs might affect chondrogenesis of the aged chondrocytes upon in vivo transplantation.