【作者】 冯爽；

【导师】 曹建平；

【作者基本信息】 苏州大学 ， 放射医学， 2006， 硕士

【摘要】 目的：探讨辐射介导的GM、ATM⁺-AT、AT细胞DNA损伤与修复；阐明毛细血管扩张性共济失调突变基因（Ataxia telangiectasia mutated，ATM）介导的乳癌基因1（breast cancer gene 1，Brca1）磷酸化及其下游DNA修复相关蛋白（DNA damage repair protein 51，RAD51）在脱氧核糖核酸（deoxyribonucleic acid，DNA）损伤修复信号传导通路中作用机理；探讨⁶⁰Coγ射线照射前、后，BRCA1、RAD51蛋白在GM、ATM⁺-AT、AT细胞中的表达。 方法：（1） 不同辐射剂量γ射线照射GM细胞、ATM⁺-AT细胞和AT细胞细胞；加入PI3K抑制剂wortmannin（WT）与对照组加入DMSO，应用碱性彗星试验（alkaline comet assay），也称碱性单细胞凝胶电泳法，测定彗星率和彗星尾长。（2） 将⁶⁰Coγ辐照后的AT细胞通过免疫共沉淀及Western Blot法分析其中ATM和BRCA1蛋白以及BRCA1和RAD51蛋白之间的相互作用。（3） 应用免疫荧光染色和激光扫描共聚焦显微镜，观察GM、ATM⁺-AT、AT细胞在照射0和10Gy⁶⁰Coγ射线后，BRCA1、RAD51蛋白在上述细胞中的定位及表达并进行定量分析。 结果：（1） 三种细胞随着辐射剂量的增大，彗星尾长逐渐增加，各个剂量组之间比较有显著差异（P＜0.001）；细胞之间比较：三种细胞尾长按GM细胞、ATM⁺-AT细胞、AT细胞的顺序逐渐增加，各个细胞组之间比较有差异（P＜0.05）；加入WT后细胞修复受到抑制，与对照组加入DMSO的尾长比较有差异（P＜0.05），且各个细胞之间比较按GM细胞、ATM⁺-AT细胞、AT细胞的顺序尾长逐渐增加，有统计学意义（P＜0.05）。（2） GM细胞、ATM⁺-AT细胞BRCA1和RAD51蛋白均有表达，而AT细胞无表达。（3） 未经⁶⁰Coγ射线照射时，BRCA1、RAD51蛋白在GM、ATM⁺-AT、AT细胞中仅有少量表达并且无共定位表达，其中在AT细胞中表达量最低；照射10Gy⁶⁰Coγ射线后，BRCA1、RAD51在GM、ATM⁺-AT、AT细胞中表达量增高，其中GM、ATM⁺-AT细胞呈现共定位表达，AT细胞无共定位表达；GM、ATM⁺-AT细胞中BRCA1、RAD51蛋白表达量与AT细胞比较均有统计学意义（P＜0.01）；GM细胞中BRCA1、RAD51蛋白表达量与ATM⁺-AT细胞比较也有统计学意义（P＜0.01）。 结论：（1） 辐射剂量在10Gy以下时随着剂量增大细胞损伤越明显；而AT细胞对辐射的敏感性最强；WT对细胞损伤后的修复有抑制作用。（2） BRCA1由ATM更多还原

【Abstract】 Objective: to investigate DNA injury and repair in GM, ATM and AT cells mediated by radiation, to investigate the mechanism for ATM mediated phosphorylation of BRCA1 and downstream gene RAD51 in signaling pathway of DNA injury repair; to investigate the expressions of BRCA1, RAD51 proteins in GM, ATM⁺-AT, AT cells before and after radiation by Coγ.Methods: （1） GM, ATM⁺-AT and AT cells treated with wortmannin （WT）, a PI3K inhibitor, or DMSO as control were radiated by y ray in different doses. Alkaline comet assay also named alkaline single gel electrophoresis assay were applied to determine comet rate and comet tail length. （2） applying immunocoprecipitation and Western blotting to analyze the interactive action between ATM and BRCA1 protein, BRCA1 and RAD51 protein （DNA repair protein RAD51） in AT cells after ⁶⁰Coγradiation.（3） the localizations and expressions of BRCA1, RAD51 proteins were detected in GM, ATM⁺-AT, AT cells by immunofluorescence staining and laser scanning confocal microscopy after 0 and 10 Gy⁶⁰Coγ radiation. Quantitative analysis was also processed.Results: （1） Comet tail length significantly increased with the doses of radiation increasing in three kinds of cells （P<0.001） and with the different cells ranking as GM cells, ATM⁺-AT cells and AT cells （P<0.05）. The comet tail length was shorter in cells treated with WT than control （P<0.05）. The comet tail length in WT treated cells increased in the order of GM cells, ATM⁺-AT cells and AT cells （P<0.05）. （2） BRCA1 and RAD51 protein were expressed in GM cells and ATM⁺-AT cells, not in AT cells.（3） Without radiation, BRCA1, RAD51 proteins were little expressed and no colocalization in GM, ATM⁺-AT, AT cells, and lowest expression were found in AT cells. After 10Gy⁶⁰Coγ radiation, the expressions of BRCA1, RAD51 proteins were increased in GM, ATM⁺-AT, AT cells. There were colocalized expressions in GM, ATM⁺-AT cells, while no in AT cells. The expressions of BRCA1, RAD51 proteins in GM, ATM⁺-AT cells significantly differentiated from the expressions in AT cells, respectively （P <0.01）.更多还原