斑马鱼Rictor基因的克

发布时间：2018-07-13 07:47

【摘要】：mTORC2隶属于PTEN/PI3K/AKT/mTOR信号通路,尤其是mTOR信号通路的过度活化与肿瘤的发生、发展密切相关,是肿瘤治疗的一个重要靶点。近年来随着临床上这类小分子抑制剂的出现,使得人们对它的兴趣越来越大,对Rictor和mTORC2相关的报道越来越多。Rictor是作为mTORC2脚手架的一个蛋白出现,共同和其他的五个蛋白维持着mTORC2的稳定。mTORC2在许多重要的生理学过程中扮演重要角色,包括：细胞生存、代谢、增殖、细胞骨架的合成等,但是相对于mTORC1来说,mTORC2有更多未知的部分需要我们去探索,比如说mTORC2的上游信号通路有哪些,它们是如何激活mTORC2的,Rictor是否维持mTORC2的稳定?在mTORC2中扮演什么角色?目前这一部分尚属空白,查阅相关文献没有确凿证据,这些都是很有趣的话题。运用斑马鱼模式生物研究Rictor的基因功能,首先我们应用生物信息学的方法,证明斑马鱼Rictor基因在进化上高度保守,为脊椎动物保守基因,为其后续在造血系统和造血微环境方面的研究提供了理论支持和铺垫。整胚胎原位杂交技术广泛运用于基因表达谱,是研究斑马鱼发育,探索相关基因的功能的重要手段。本研究根据斑马鱼rictor mRNA设计构建合成反义RNA探针,整胚胎原位杂交技术分析了rictor在斑马鱼早期发育过程中(时间和空间上)的表达情况,Zrictor在胚胎的单胞期就开始表达,24小时特异性的表达于脊索,48到72小时特异性的表达与神经管和肌节。通过反义吗啉代寡核苷酸MO(低表达)抑制Zrictor后,结果发现mo注射组斑马鱼胚胎出现很少量的心包积液和身体弯曲(主要在尾部),并且在发育早期呈现一定的发育滞后。显微注射前后斑马鱼时空表达的差异,在血液系统的marker和心脏marker里面没有出现明显的变化,但是Fli-1在节间血管有轻微变化,肌肉的表达(myod)有所减少；有心包积液的斑马鱼胚胎用o-staining检测循环形成后斑马鱼的血红蛋白的分布表达变化,显示血红蛋白的沉积减少。在提倡小分子抑制剂只对肿瘤起效,而不影响正常组织功能的情况下,Rictor是否是合适的打靶对象?我们在体内验证了rictor被MO抑制后对正常组织和造血系统的影响,可能为临床血液病和肿瘤治疗的新靶点。
[Abstract]:MTORC2 belongs to the PTEN / PI3K / AKT / mTOR signaling pathway, especially the overexpression of mTOR signaling pathway is closely related to the occurrence and development of tumor, and is an important target of tumor therapy. In recent years, with the emergence of such small molecular inhibitors in clinic, people are more and more interested in it. More and more reports about Rictor and mTORC2. Rictor is a protein of mTORC2 scaffold. The common and other five proteins maintain the stability of mTORC2. MTORC2 plays an important role in many important physiological processes, including cell survival, metabolism, proliferation, cytoskeleton synthesis, etc. But there are more unknown parts of mTORC2 than mTORC1. For example, what are the upstream signaling pathways of mTORC2, and how do they activate the Rictor of mTORC2 to maintain the stability of mTORC2? What role do you play in mTORC2? At present, this part is still blank, there is no conclusive evidence to consult the relevant literature, these are very interesting topics. Using zebrafish model organisms to study the gene function of Rictor, we first use bioinformatics method to prove that the zebrafish Rictor gene is highly conserved in evolution and belongs to vertebrate conservative gene. It provides theoretical support and foreshadowing for the further study of hematopoietic system and hematopoietic microenvironment. Whole embryo in situ hybridization is widely used in gene expression profiling, which is an important means to study the development of zebrafish and explore the function of related genes. In this study, antisense rictor probes were designed and synthesized from zebrafish. The expression of rictor during the early development of zebrafish (in time and space) was analyzed by in situ hybridization of whole embryo. The expression of nerve tube and muscle ganglion. After inhibition of Zrictor by antisense morpholine oligonucleotide (MO), it was found that a small amount of pericardial effusion and body curvature (mainly in the tail) appeared in the embryos of zebrafish in mo injection group, and showed a certain developmental retardation at the early stage of development. Before and after microinjection, there were no significant changes in marker and cardiac marker in the blood system, but there was a slight change of Fli-1 in internode vessels and a decrease in the expression of (myod) in muscle. The distribution and expression of hemoglobin in zebrafish embryos with pericardial effusion were detected by o-staining. Is Rictor an appropriate target when it is advocated that small molecular inhibitors work only on tumors without affecting normal tissue function? We have confirmed in vivo the effect of rictor suppression by MO on normal tissue and hematopoietic system, which may be a new target for clinical therapy of hematologic diseases and tumors.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R-331

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