DAB2IP基因对电离辐射后DNA损伤应答修复的影响与作用机制研究

DAB2IP是新发现的抑癌基因，ATM和DNA-PK是DNA损伤应答（DDR）通路的关键蛋白，分别启动同源重组修复(HR)和非同源末端连接(NHEJ)过程。课题拟从细胞、动物和临床三个层面讨论DAB2IP与电离辐射后DDR通路的时空关联：1）以RNA干扰和目的基因转染技术双向调节DAB2IP表达，建立细胞和动物模型；2）γ射线照射后WesternBlot（WB）检测ATM和DNA-PK及磷酸化蛋白，以KU-55933和NU7026分别抑制ATM和DNA-PK，集落和生长曲线法评价模型细胞和动物的放射敏感性；3）KU-55933联合γ射线，WB观察ATM下游效应蛋白（KAP-1等及磷酸化产物），免疫荧光检测RAD51 foci，推测DAB2IP对ATM依赖的HR过程影响；4）收集正常上皮、良性增生和癌组织标本，免疫组化分析DAB2IP同放疗预后的相关性，加深了解DAB2IP的放射生物学功能。

DAB2IP is a newly reported tumor suppressor gene. ATM and DNA-PK are the key proteins of radiation-induced DNA damage repair pathway (DDR), respectively, to start homologous recombination repair (HR) and non-homologous end joining (NHEJ) . In this study, the temporal and spatial relationship between DAB2IP and DDR post ionizing radiation (IR) will be discussed on three levels: in vitro, in vivo and clinical specimens: 1) establishing cell and animal models by RNA interference and the target gene transfection technique to decrease and increase DAB2IP expression, respectively. 2) Detecting ATM and DNA-PK and their phosphorylated products under γ-ray of 2Gy with Western Blot (WB). KU-55933 and NU7026 are used to inhibit ATM and DNA-PK activity, colony formation assay and growth curve analysis are applied to evaluate in vitro and in vivo sensitivity to IR. 3) When treated with KU-55933+IR, ATM downstream effector proteins (KAP-1, etc., and the phosphorylated products) and RAD51 foci are detected by WB and immunofluorescence assay. Discussing the effect of DAB2IP on ATM-dependent HR pathway. 4) Collecting specimens of the normal epithelium, benign hyperplasia and cancer tissue, DAB2IP expression are determined by immunohistochemical analysis. Combined with patient history information, the correlation between DAB2IP and the prognosis of radiotherapy are speculated to better understanding the radiobiological features of DAB2IP.