细胞衰老相关蛋白PTRF的鉴定及其功能分析摘　要细胞衰老是在其内部或外部因素的诱导下，通过细胞内一系列蛋白因子的信号传导作用来实现的生物学过程。介导细胞走向衰老的信号传导通路主要有两条：p53通路、Rb通路。目前为止，对于两条信号通路的上下游调控因子及其功能尚不清楚，有待于进一步研究。本研究在前期蛋白质组学所筛选的细胞衰老相关蛋白质中，选取部分在年轻细胞与衰老细胞中表达差异显著的蛋白质，进行进一步的筛选、鉴定。根据鉴定结果，选取有代表性的RNA聚合酶Ⅰ转录释放因子PTRF（polymeraseⅠand transcript-release factor），对其进行细胞定位分析和功能研究。我们以人胚肺成纤维细胞系WI38和IMR90为衰老模型。研究发现，在两种细胞系中，随着细胞的增殖，PTRF的表达量逐渐增多；同时，PTRF的细胞定位也发生变化。在年轻细胞中，PTRF主要定位于细胞核内，而随着细胞的增殖，蛋白的定位逐渐由细胞核内转为细胞膜上，在细胞膜上呈不均匀分布，这一分布与胞膜窖caveolae结构定位相吻合。PTRF蛋白在其36、40、365、366位丝氨酸存在磷酸化位点，我们利用定点突变的方法，研究其磷酸化位点对蛋白定位及其功能的影响。研究发现，PTRF的入核与其N端磷酸化相关，当36、40位丝氨酸单突变或全突变时，PTRF的细胞定位受到影响，导致蛋白在年轻细胞中不能进入细胞核。在年轻WI38细胞中过表达PTRF之后，细胞增殖受到抑制，细胞寿命明显缩短。综上，PTRF通过其表达量和定位的改变使其在细胞中的作用发生变化，这种变化对细胞的衰老起到了促进作用。关键词：PTRF，细胞衰老，定位，caveolae，突变，过表达

IDENTIFICATION AND FUNCTIONAL ANALYSIS ON CELLULAR SENESCENCE ASSOCIATED PROTEIN PTRFABSTRACTCellular senescence is a program induced by intrinsic or extrinsic stresses and finally achieved by the interaction of a series of proteins involved in the cellular senescence signal transduction.The two paradigmatic tumor suppressor proteins, p53 and Rb, have been shown to play critical roles in the signal transduction of cellular senescence. Nevertheless, the target genes and pathways of upstream and downstream p53 and Rb remained unclearly.In previous study, numbers of cellular senescence associated proteins have been identified through proteomic profiling approach, isobaric tagging for relative and absolute quantitation (iTRAQ). Result from proteomic profiling were validated by RT-PCR and western blotting for selected protein PTRF（polymeraseⅠand transcript-release factor），on which was ultimately focused in the further study .Using human embryo lung fibroblast cell lines WI38 and IMR90 as the cellular senescence model, we found that expression of PTRF gradually increased in protein level but not transcription level in WI38 and IMR90 during senescence; and the subcellular localization of PTRF has experienced dramatically changes. PTRF was mainly localized in nucleus in young fibroblast, while it was mostly localized in the cell membrane in senescent fibroblast. It was interesting that PTRF and caveolae were co-localized in the fibroblast cell membrane.There were four phosphorylation sites in PTRF which were localized to Ser-36, Ser-40, Ser-365 and Ser-366. In this work, the effect of phosphorylation sites on the protein subcellular localization and protein function was investigated by the site directed mutagenesis. It was found that the nucleus localization of PTRF was associated with the protein N terminal phosphorylation sites. When the Ser-36, Ser-40 was or were mutated, PTRF could not localize in the nucleus in young fibroblast cells. And consistent with our expectation, overexpression of PTRF in young fibroblast resulted in reducing the cell life span, compared with control.To sum up, PTRF plays an important role in the fibroblast cellular senescence through increasing expression and changing in subcellular distribution.KEY WORDS：PTRF, cellular senescence, subcellular localization, caveolae，mutagenesis, overexpression