一种辐射诱导肠上皮表达基因的分子克隆与鉴定

Intestinal epithelium is sensitive to the radiation, severe damage of which can cause the fatal radiation gastrointestinal syndrome. Our project focus on the gene mechanism of the intestinal epithelium's sensitivity to the radiation so as to elucidate the molecular mechanism of the damage and repair of the irradiated intestinal epithelium and promote the repair of the intestinal epithelium..The Different Display of mRNA was employed to analysis the expression profile of the mouse intestinal epithelium before and after radiation. The differently expressed gene fragment RS1 was cloned successfully and identified. There was no significant homologous sequence was found in the GENE BANK which hint it is a radiation induced new gene. RT-PCR and in situ hybridyzation were used to identified RS1 expression in different tissues such intestine, liver, kidney, spleen and myocardium. All of these indicate the important potential function of RS1.In the other hand some works covered different molecular levels were applied to expand the research on target genes related to the damage and repair induced by radiation. A different expressed genes subtracted cDNA library of 35Gy γray irradiated IEC-6 cells and normal counterpart is successfully constructed with SSH and T/A clone techniques. About 14 radiation induced ESTs related to cell proliferation,signal transduction,cell skeleton and stress response ect were cloned and identified. we have comparatively analysed the proteome of IEC-6 cell line as an in vitro model and mouse intestinal epithelial cells as an in vivo model with its irradiated counterpart, respectively. A series of methods, including 2-D electrophoresis, were used to separate and identify the differential proteins. Image analysis revealed that averages of 608±39 and 595±31 protein spots were detected in normal IEC-6 cells and irradiated IEC-6 cells, respectively. 32 protein spots showed significant expression alteration in irradiated IEC-6 cells compared with that of normal cells. Also 638±39 and 566±32 protein spots were detected in normal mouse enterocytes and irradiated counterparts, and 36 protein spots were significantly altered. Thirty-six differential protein spots were isolated from gels, digested with trypsin, and measured with MALDI-TOF-MS. A total of 33 spots yielded good spectra, and 28 spots matched known proteins after database searching. These proteins were mainly involved in anti-oxidation, metabolism, signal transduction, and protein posttranslational processes. Some proteins display the similar variation in in vivo and in vitro model which indicate the radiobiology characteristic worthy of research..In summary, the different pathological changes of the intestinal epithelia during injury state and regeneration state were observed and analysed through different advanced technics in different molecular level. A couples of related genes have been identified and some work have been done about RS1. All of these present a promising way to elucidate the molecular mechanism of the damage and repair of the irradiated intestinal epithelium and promote the repair of the intestinal epithelium. The similar reports have not been searched in the domestic and foreign journals. Seven papers have been finished.

在辐射损伤的小鼠肠上皮细胞中应用mRNA差异显示技术克隆到一条特异表达基因片段，初步证实为一种新型肠粘蛋白。在此基础上，本项目拟采用RACE、基因测序、体外转录与翻译分析等技术，扩增出该基因的全长序列，对其进行结构、功能分析及染色体定位。使人们对该基因有一个全新的认识，并为在基因水平深入探讨辐射致肠道损伤机理提供实践依据。