Rheb在急性肠炎中的功能研究

发布时间：2018-05-02 22:36

本文选题：Rheb + mTOR信号通路　；参考：《华东师范大学》2014年硕士论文

【摘要】：mTOR信号通路通过感知胞外营养状况指导胞内蛋白质合成,调节细胞生长、增殖与存活,在生理和病理过程中起着非常重要的作用。Rheb是一个小GTP酶,它作为mTOR的上游激活蛋白,Rheb-GTP通过直接与mTOR结合激活1mTORC1复合物从而开启mTORC1信号通路。利用基因敲除小鼠,研究者发现Rheb在早期胚胎发育和神经系统发育中起着重要的作用,但是其在其他系统发育和病理过程中的作用还不清楚,在肠急性损伤修复中的作用还没有相关研究。我们购买Rheb基因突变ES细胞,通过显微注射构建了Rheb基因敲除小鼠。由于Rheb纯合突变小鼠胚胎致死,我们利用Rheb杂合突变小鼠展开研究。通过葡聚糖硫酸酯(DSS)诱导的急性肠炎模型研究了Rheb参与调节的mTORC1信号通路在肠急性损伤修复中的功能。实验发现,Rheb+/-小鼠的肠发育和组织结构没有明显缺陷,但是在DSS诱导急性肠炎后,与野生型小鼠相比,Rheb+/-小鼠的病征更加明显,肠上皮细胞出现更大面积的溃疡,肠上皮细胞增殖减少、凋亡增多,在DSS诱导10天左右全部死亡,而野生型小鼠绝大部分都能存活。由于肠炎模型牵涉到肠组织和免疫系统两个靶组织,而研究所用的小鼠为全身突变的品系。为了研究肠炎过程中Rheb在哪个系统中的功能更为重要,我们采取骨髓移植的模型。通过骨髓移植实验发现Rheb突变的小鼠不管是接受野生型小鼠还是突变型小鼠的骨髓,肠炎表型都非常剧烈；而野生型小鼠不管接受野生型还是突变型小鼠的骨髓,炎症程度都比较轻微。说明Rheb主要是在非免疫系统中参与调解肠炎的发展进程。通过检测mTORC1下游蛋白的变化,证明Rheb是通过mTORC1信号通路对肠炎起应答反应的。给小鼠体内注射氨基酸以激活mTORC1信号通路,Rheb+/-小鼠的肠炎得到一定的缓解。综上所述,DSS诱导的急性肠炎模型中,Rheb/mTORC1是对肠上皮细胞起保护作用。由于mTOR抑制剂雷帕霉素类似物作为抗癌药物已在临床上应用,本研究为该类抗癌药物是否适用于患有急性肠炎的癌症患者提供重要的参考。
[Abstract]:MTOR signaling pathway plays a very important role in physiological and pathological processes by sensing extracellular nutrition to guide intracellular protein synthesis, regulate cell growth, proliferation and survival. Rheb is a small GTP enzyme. It acts as the upstream activating protein of mTOR, Rheb-GTP, and activates the 1mTORC1 complex by directly binding with mTOR to activate the mTORC1 signaling pathway. Using gene knockout mice, the researchers found that Rheb plays an important role in early embryonic and nervous system development, but its role in other phylogenetic and pathological processes remains unclear. No studies have been conducted on the role of these agents in the repair of acute intestinal injury. We purchased es cells with Rheb gene mutation and constructed Rheb knockout mice by microinjection. Since Rheb homozygous mutant mice died from embryo, we used Rheb heterozygous mutant mice to study. An acute enteritis model induced by dextran sulfate (DSS) was used to study the function of mTORC1 signaling pathway regulated by Rheb in the repair of acute intestinal injury. It was found that there were no obvious defects in intestinal development and tissue structure in Rheb / R mice, but after acute enteritis induced by DSS, the symptoms of Rheb /-mice were more obvious than those of wild type mice, and the intestinal epithelial cells had larger ulcers. The proliferation and apoptosis of intestinal epithelial cells decreased and all of them died at 10 days after induction by DSS, while most of the wild type mice survived. Because the enteritis model involves two target tissues, the intestinal tissue and the immune system, the mice used in the study are systemic mutant strains. In order to study the function of Rheb in which system is more important during enteritis, we adopted the model of bone marrow transplantation. The bone marrow transplantation test showed that the enteritis phenotype of the mice with Rheb mutation was very intense, whether it was the wild type mice or the mutant mice, while the wild type mice accepted the bone marrow of the wild type mice or the mutant mice. The degree of inflammation is mild. Rheb is mainly involved in mediating the development of enteritis in the non-immune system. By detecting the changes of downstream protein of mTORC1, it is proved that Rheb responds to enteritis through mTORC1 signaling pathway. The enteritis in mice was alleviated by injecting amino acids into mice to activate the mTORC1 signaling pathway. In conclusion, Rheb / mTORC1 plays a protective role on intestinal epithelial cells in the model of acute enteritis induced by DSS. As the mTOR inhibitor rapamycin analogue has been used as an anticancer drug in clinic, this study provides an important reference for the application of this kind of anticancer drugs to cancer patients with acute enteritis.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574

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