HMF3A_with_3Dwt

HMF3A cells, created from adult human mammary fibroblasts by immortalisation with a thermolabile SV40 large T antigen and the catalytic sub-unit of human telomerase, undergo co-ordinated induction of cellular senescence upon inactivation of T antigen. HMF3A cells cease proliferating by 4 days at 39°C. We directly compared the gene expression profiles of cells at 33.5°C and after shift up to 39.5°C for 7 days (10 arrays, 3 biological replicates). To eliminate genes that change in response to the temperature shift, we compared the profiles of HMF3Dwt cells at 33°C and those shifted up to 39°C for 7 days (4 arrays, 2 biological replicates). Since HMF3Dwt cells were immortalised with wild type U19 LT antigen, they proliferate at both temperatures. To break up the list of genes obtained with the 7 day shift, we performed several further comparisons on the arrays. HMF3A cells at 33°C were compared to the primary donor fibroblasts (HMF3 passage 8, 4 arrays), HMF3A cells shifted to 39°C for 2 days (4 arrays, 2 biological repeats), HMF3A cells shifted to 39°C for 7 days and then shifted back to 33°C for 3 days (8 arrays, 4 biological repeats) or 7 days (4 arrays, 2 biological repeats). As a comparison to the irreversible process of senescence, we sought to identify genes that changed upon reversible growth arrest, quiescence. Thus changes that occurred when the cells were grown to confluency and starved were also determined (2 biological replicate, 6 arrays). Keywords = Rb Keywords = fibroblast Keywords = immortalization Keywords = LT antigen Keywords = p53 Keywords = quiescence Keywords = senescence Keywords: ordered

HMF3A细胞是通过将热不稳定SV40大T抗原（thermolabile SV40 large T antigen）与人端粒酶催化亚基（catalytic sub-unit of human telomerase）导入成人乳腺成纤维细胞并进行永生化构建得到的，该细胞在T抗原失活后会发生协同诱导的细胞衰老（cellular senescence）。HMF3A细胞在39℃条件下培养至第4天时即停止增殖。
本研究直接比较了33.5℃下培养的细胞，与升温至39.5℃并培养7天后的细胞的基因表达谱（共10张基因芯片，3次生物学重复）。为排除仅由温度转换引发的基因表达变化，研究团队还比对了33℃下的HMF3Dwt细胞与升温至39℃并培养7天后的该类细胞的基因表达谱（共4张基因芯片，2次生物学重复）——HMF3Dwt细胞经野生型U19 LT抗原永生化，因此在两种温度下均可正常增殖。
为梳理7天温度转换后获得的基因列表，研究团队开展了多组额外的芯片比对实验：将33℃下的HMF3A细胞分别与原代供体成纤维细胞（HMF3第8代，共4张芯片）、升温至39℃并培养2天的HMF3A细胞（共4张芯片，2次生物学重复）、升温至39℃并培养7天后再回移至33℃分别培养3天（共8张芯片，4次生物学重复）或7天（共4张芯片，2次生物学重复）的HMF3A细胞进行比对。
为与不可逆的衰老过程形成对照，研究团队还试图鉴定可逆性生长阻滞即静息（quiescence）状态下的差异表达基因，因此也检测了细胞达到汇合状态并进行饥饿培养时的基因表达变化（2次生物学重复，共6张芯片）。
关键词：Rb、成纤维细胞、永生化、LT抗原、p53、静息、衰老、有序