γ射线照射前后IEC-6细胞蛋白质组的差异分析  

Differential proteomic analysis of intestinal epithelium cell line before and after irradiation by gamma-ray

作　　者：章波[1] 粟永萍[1] 艾国平[1] 刘晓宏[1] 魏永江[1] 王锋超[1] 程天民[1] 

机构地区：[1]第三军医大学预防医学院防原医学教研室,全军复合伤研究所,重庆400038

出　　处：《第三军医大学学报》2003年第24期2157-2160,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金资助重点项目 ( 30 2 30 36 0 ;39970 2 32 )~~

摘　　要：目的　分析γ射线照射后肠上皮细胞系 (IEC 6)细胞蛋白质组的改变。方法　分别提取正常IEC 6细胞和经过 2 5Gy照射后细胞的蛋白样品 ,采用固相pH梯度双向凝胶电泳技术进行分离 ,考马斯亮蓝染色后经PDQuest软件分析双向电泳图谱 ,对部分差异的蛋白点进行胶内酶切、基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)测定其肽质量指纹图谱 ,在网上检索数据库鉴定差异蛋白 ,并采用RT PCR间接证实其变化。结果　获得了较好的双向电泳图谱。图像分析显示 :在正常IEC 6细胞中检测到 ( 60 8± 3 9)个蛋白质点 ,细胞照射后的样品中检测到 ( 5 95± 3 1)个蛋白质点 ,其中匹配的蛋白点为 3 96个。对表达差异的 16个蛋白质点进行肽质量指纹图谱分析 ,经数据库检索后初步鉴定为一些与代谢、自由基清除、细胞骨架相关的蛋白。RT PCR证实了ERP2 9基因在照射后增强。结论　电离辐射可以诱导IEC 6细胞蛋白质组发生改变。Objective To identify the proteins associated with ionizing irradiation of intestinal epithelium cell line (IEC 6), and to elucidate the molecular mechanism of reconstruction of intestinal epithelium damaged by ionizing irradiation. Methods Protein samples extracted from normal IEC 6 cells and ICE 6 cells irradiated by γ ray at the dose of 25 Gy were separated by immobilized pH gradient two dimensional (2 D) gel electrophoresis. After Coomasie blue staining, the 2 D electrophoretogram was analyzed using PDQuest software. The differential proteins were cut off from the Coomasie blue stained gel and digested in gel with trypsin, and then identified by peptide mass fingerprinting based on matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) and database searching. Total RNA of IEC 6 cells was isolated and RT PCR analysis was performed using primers of ERP29 and GAPDH. Results After Coomasie blue staining, the 2 DE image analysis by PDQuest software detected (608±39) spots in normal IEC 6 cell sample and (595±31) spots in irradiated cells, of which 396 spots were matched. Sixteen differential spots were cut off from the Coomasie blue stained gel, and 11 spots were preliminarily identified. These proteins were associated with cell metabolism, cytoskeleton, and free radical elimination, etc . Ionizing radiation induced mRNA of ERP29 in a time dependent manner over a 48 h period. Conclusion γ ray irradiation can induce changes of the protein expression pattern of intestinal epithelium cell line. This helps to understand the molecular mechanisms of ionizing radiation on intestinal epithelium cells.

关 键 词：电离辐射 肠上皮细胞 双向电泳 肽质量指纹图谱分析 

分 类 号：R394.3[医药卫生—医学遗传学] Q345[医药卫生—基础医学]