消减杂交筛选2BS细胞衰老过程中差异表达基因

细胞的复制性衰老最终导致不可逆的G1 期阻滞 ,研究此过程中差异表达基因对于阐明衰老发生机制有重要意义 .分别构建年轻和衰老 2BS细胞高表达基因的消减文库 ,经点杂交筛选后共得5 3个差异表达基因 .对其中部分基因的VirtualNorthern印迹分析证实差异表达确实存在 .选择Y1 1 4和S1 1 1片段 ,以Northern印迹分析确证其表达变化 ;并通过对新生儿和老年人白细胞中二者的表达分析 ,显示二者在体内也存在与体外衰老过程相一致的随增龄表达变化 .结果在一定程度上体现了 2BS细胞衰老过程中基因表达谱的变化 ;首次报道了TSSC3(tumorsuppressingsubtransferablecandidate 3)、hnRNPK (heterogeneousnuclearribonucleoproteinK)等基因在成纤维细胞衰老时发生差异表达 ;通过对Y1 1 4和S1 1 1在体内衰老时的表达分析 ,显示体内和体外衰老有一定的相关性

Screening for Differentially Expressed Genes upon 2BS Fibroblasts Senescence by Subtractive Hybridization

Replicative senescence causes an irreversible G 1 arrest finally. The study of differentially expressed genes upon senescence is important to clarify the mechanisms underlying the process. Construction and differential screening of subtracted cDNA libraries enriched with cDNAs were accumulated in young and senescent 2BS fibroblasts respectively,and resulted in the cloning of 53 differentially expressed gene fragments. The virtual Northern blot analyses of some fragments verified the differential expression. The differential expression of two cDNA fragments Y114 and S111 were confirmed by Northern blot analyses. The semi quantitative analyses of expression of these two fragments in blood leukocytes from old and new born donors showed that the trends of the changes were consistent with those in cultured 2BS cells. The changes of gene expression profile upon the senescence of 2BS fibroblasts were manifested to a certain extent. The differential expression upon senescence of TSSC3 (Tumor suppressing subtransferable candidate 3) and hnRNP K (Heterogeneous nuclear ribonucleoprotein K) was observed for the first time, and the relationship between replicative senescence and organismal aging was supported by the consistent trends of expression changes in leukocytes in vivo and 2BS fibroblasts in vitro .