p73在Hutchinson-Gilford Progeria Syndrome 中对DNA损伤修复通路调控的机制研究

HGPS is a premature aging disease which is generally considered as a model of the normal aging process. It has been confirmed that the abnormal DNA damage repair pathways causes genome instability, and ultimately leads to cell senescence. p73 is a member of the p53 family with the different signaling pathways. Studies show that in the normal human cells, p73 expression is very low, however, in many tumor cells p73 is highly expressed. The latest study found that p73 regulated DNA damage and repair pathway by affecting DNA damage checkpoint, but the detailed mechanism is unclear. Based on previous work, we found that p73 gene expression was significantly up-regulated in the HGPS cells with high passage number by using PCR array. In this project, we will build up a HGPS cell line with p73 silencing, then measure the cell cycle,apoptosis,senescence and DNA damage.And we will use the UVC to cause the DNA damage in normal fibroblasts with over expression p73.We will use chromatin-immunoprecipitation on microarray to detect the target genes which involved in DNA damage and repair pathway and regulated by p73. And the Co-immunoprecipitation will be preformed to detect the protein-protein interaction of P73 DNA damage and repair pathway in HGPS cells. The aims of this project are to find out the network of p73 regulation on DNA damage and repair pathway in the process of aging and strive to provide a new target for clinical treatment.

HGPS是过早老化疾病中的一种典型病例，被普遍认为是研究正常衰老进程的一个理想模型。研究已证实, DNA损伤修复通路异常是造成基因组不稳定，最终引起细胞老化的重要原因。p73是p53家族成员，但与p53作用的信号途径有所不同。研究显示在人正常细胞中p73表达极低，而在许多肿瘤细胞中p73又是高表达的。最新研究发现，p73在DNA损伤修复通路中可调控细胞周期检查点，但其详细的作用机制尚不清楚。在前期工作基础上，我们运用基因芯片发现p73基因在衰老HGPS细胞里表达显著上调。本项目拟构建p73沉默的HGPS细胞株,检测其细胞周期、细胞衰老、细胞凋亡及DNA损伤情况。同时对p73高表达的正常成纤维细胞进行DNA损伤刺激，然后利用染色质免疫沉淀方法结合芯片筛选出受p73调控的参与DNA损伤修复靶基因，并用免疫共沉淀和免疫荧光的方法研究P73在HGPS细胞中与参与DNA损伤修复蛋白的相互作用机制。

HGPS 是过早老化疾病中的一种典型病例，被普遍认为是研究正常衰老进程的一个理想模型。研究已证实, DNA 损伤修复通路异常是造成基因组不稳定，最终引起细胞老化的重要原因。p73 是p53 家族成员，但与p53 作用的信号途径有所不同。最新研究发现，p73 在DNA损伤修复通路中可调控细胞周期检查点，但其详细的作用机制尚不清楚。我们前期工作运用基因芯片发现p73 基因在高代数HGPS 细胞里表达显著上调。本课题首先以低，中，高代数的HGPS 细胞作为研究对象，在基因及蛋白水平验证了p73的变化，结果与芯片相符。其次发现p73与细胞周期有关，且对DNA双链损伤有促进作用。接下来我们研究p73参与DNA损伤修复途径的机制发现：在HGPS细胞中抑制p73的表达可以下调RPA和上调PCNA。然后我们运用染色质免疫共沉淀结合启动子芯片的方法筛选出p73的靶基因参与了的信号通路，最后分析发现p73在HGPS细胞发生了DNA损伤后作为转录因子调节了如MUTYH,RPA等靶基因的表达从而延缓了DNA的修复，影响了衰老的进程。