摘要
目的:探讨体外培养猪耳软骨细胞在老化过程中,软骨蛋白聚糖(aggrecan)的代谢状况与分子结构的变化及其水解酶(aggrecanase)表达水平的改变。由此进一步阐明aggrecan在组织工程软骨构建中起决定性的作用。方法:取体外培养P1-P8各代猪耳软骨细胞及其培养液,爱茜蓝(Alcian Blue)法检测培养液中蛋白聚糖的含量和糖胺聚糖(glycosaminoglycan,GAG)的分子结构。RT-PCR检测aggre-canase-1和-2的mRNA表达水平。结果:在P1细胞的培养液中,蛋白聚糖的含量和长链/高度硫酸化的GAG含量都是最高的。由P4细胞开始,这2项指标均显著减少(P<0.01),且两者的变化趋势有显著的相关性(P<0.01)。Aggrecanase-1 mRNA在前3代细胞基本无表达,P4细胞中有显著上升(P<0.01),随后逐渐减少,P8细胞又不再表达。Aggrecanase-2 mRNA的表达呈随机状态,与细胞代数无显著性相关(P>0.05)。结论:体外培养软骨细胞的老化对aggrecan的代谢有非常大的影响。一方面aggrecan的合成减少、大分子聚合物形成受阻;另一方面胞外基质的水解作用加剧,最终导致老化细胞的基质崩解。
AIM: To investigate the changes in catabolism and molecular structure of aggiecan and in expression of aggrecanases during aging of in vitro cultured chondrocytes. METHODS: The medium from PI to Pg porcine chondro-cytes was harvested and lyophilized. Alcian Blue precipitation was utilized for quantification of proteoglycan and structural analysis of glycosamino-glycans (GAGs). The expression of mRNA encoding aggrecanase-1 and -2 was measured by RT-PCR. RESULTS: The amount of proteoglycan and longer chain/higher sulfated GAGs was higher in the medium of P1 cells than in the medium of other passage cells. They began to reduce significantly in P4 cells ( P < 0.01) . There was significant correlation between the changes of proteoglycans and the contents of longer chain/higher sulfated GAGs( P < 0. 01). There was no expression of aggrecanase-1 in P1 to P3 chondrocytes. Its expression level, however, increased transitionally in P4 chondrocytes ( P < 0. 01) and decreased stepwise. The expression of aggrecanase-2 was in a random pattern, which was not correlated with cell aging(P>0. 05) . CONCLUSION: The catabolism of aggrecan was greatly affected by the aging of chondrocytes cultured in Vitro. The decreased synthesis and aggregation of aggrecan and the increased proteolysis led to the degradation of extracellular matrix.
出处
《中国临床康复》
CSCD
2003年第2期216-217,T002,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家"973"组织工程研究项目04项子课题(G1999054304)