DNA微阵矩分析人软骨细胞体外老化过程中基因表达水平的变化

Microarray analysis of age-related gene expression of human chondrocytes cultured in vitro

目的研究成人软骨细胞体外单层培养老化过程中基因表达水平的变化,初步探索人软骨细胞老化发生的可能机制。方法取体外培养的第1代(P1)至第4代(P4)人肋软骨细胞,观察细胞增殖率,阿利新兰(Alcianblue)法定量检测硫酸软骨素蛋白聚糖聚集体(aggrecan)中糖胺多糖(GAGs)含量,RT-PCR分析Ⅰ、Ⅱ型胶原及aggrecan基因表达量,以观察人软骨细胞体外老化过程。并采用DNA微阵矩技术对P1及P4软骨细胞表达差异的基因进行比较分析,P1及P4人肋软骨细胞基因表达水平差异≥2倍时,被认为差异具有显著性。结果P4软骨细胞从细胞增殖率,细胞中GAGs含量,Ⅱ型胶原及aggrecan基因表达量分析均明显低于P1软骨细胞(P<0.01)。在1000点的含有原癌基因、抑癌基因、细胞凋亡、应激反应蛋白基因和免疫相关基因的芯片分析中,P1及P4间存在差异的基因共36个,其中在P4中下调的基因11个,主要为细胞外基质、生长因子转录因子等,而在P4中上升的基因共25个主要为蛋白酶类、炎症因子及凋亡相关基因等。结论P4软骨细胞呈现老化特征。通过DNA微阵矩技术发现P4老化细胞中基质合成相关基因表达下降,而降解酶类基因表达上升,致细胞外基质减少;且细胞增殖有关基因表达的减少及凋亡因子的增加,老化细胞增殖率下降。

Objective Chondrocytes become aged easily dur ing in vitro expansion,and are unabl e to form cartilage upon in vivo transplantation.This study is an attempt to explore aging mechanism of in vivo cultured human chondrocytes by microarray analysis of gene expression changes.Methods The spare human costal cartilage fro m ear reconstruction pa-tients(n =3,aged 10-20)was digested with collagenase II to i solate human chondrocytes(HCC)which were to be expanded from P 1 to P 4 to study cell aging.Cell proliferation and the amount of glycosaminogly cans(GAGs by Alcian Blue precipita-tion)were examined from P 1 to P 4 .The gene expressions of collagensⅠandⅡ,and aggrecan were semi-quantified by RT-PCR.For microarray,RNA was isolated from the P 1 and P 4 cells and labeled with cy3and cy5flu orescence respectively as probes to hybridize a cDNA microarray(about 1000genes).Those genes with 2-fold difference of expression were se-lected.Results From P 1 to P 4 ,HCCs changed their morphology from polygon to fibroblast-like shape,d ecreased their cell proliferation by 46.8%(P<0.01),typeⅡcollage gene expression by 25%(P<0.05),and aggrecan gene expression by 12%(P<0.05).Contrarily,the typeⅠgene expression increased by 18%fro m P1to P 4 (P<0.05),indicating that P 4 were truly aged cells.Microarray re sults showed that 11genes were down-regulated at P 4 ,including extracellular matrices,growth factors,and transcription f actor genes.The up-regulated 25gen es at P 4 included proteases,inflammatory f actors and apoptosis genes.Conclusions P 4 chondrocytes are revealed to be aged phenotypes.The down-regulation of anabolic metabolism related genes and up-reg ulation of proteases genes lead to extra cellular matrix decrease.Decre ased production of growth factors and increased apopto sis may cause decreased cell prolife ration of aged chondrocytes.[