stil基因在PC12细胞和斑马鱼视网膜细胞增殖中的作用及斑马鱼帕金森疾病模型的建立

发布时间：2018-04-29 21:35

本文选题：多巴胺细胞 + 6-羟多巴　；参考：《南开大学》2014年博士论文

【摘要】：细胞增殖普遍存在于自然界,从低等的动植物的细胞分裂到高级生物的生长发育都离不开细胞增殖,而且细胞增殖在生物体的生长发育过程中受到非常严谨而又精确的调控,一旦调控网络中的某部分功能改变,则细胞增殖就会被打乱,进而影响生物体的正常生长发育或生理功能的实现。组织与器官的再生离不开细胞增殖,组织器官的再生与修复都是要通过细胞不断增殖分裂实现的。当生物体的某部分组织或器官受到损伤后,就会启动相应的信号通路来调控并实现组织和器官的修复。在自然界众多物种中,低等生物显示出比高等生物更强的再生能力,比如水螅、涡虫、甚至非洲爪蟾、鱼类、蝾螈等,而作为高等生物的哺乳类则在漫长的进化中丢失了某些再生能力。本实验中采用的斑马鱼既具有很强的再生能力,而且其基因组还与人类基因组存在很高的同源性,非常具有研究意义。 Stil (SCL/TAL1interrupting locus)基因,最早被发现于急性T细胞淋巴白血病人中,随着研究的不断深入,还发现当该基因发生突变时,会导致人的先天性小头畸形症,而且还通过与Shh信号通路起作用调控小鼠的左右对称发育。随后研究学者在很多癌症细胞中发现该基因上调表达,进一步研究发现Stil基因的表达产物是中心体的组成部分,在中心体的组装和复制中起着重要作用,当缺失Stil基因时,细胞中会缺失中心体；而当过表达Stil基因时,细胞中则会出现2个甚至多个中心体。本实验首先以PC12神经细胞模型为研究对象,发现STIL蛋白在PC12细胞中的定位不仅存在细胞质,还存在细胞核内,暗示着Stil基因可能还有别的未知功能；我们还证实了PC12细胞中的STIL也可以与SUFU和GLI1发生相互作用,而且还可能存在异源三聚体的蛋白复合体形式；当沉默Stil基因时,细胞的增殖速度减慢,而且Shh信号通路的标志基因glil的表达也下调了；过表达Stil基因时,PC12细胞的增殖速度加快,glil的表达也升高,其靶基因cyclinD2的表达也升高了,至此我们推测Stil基因可以通过调控glil基因来间接调控cyclinD2的表达,进而影响周期蛋白依赖性激酶CDK的活性,从而实现对细胞周期的调控。另外,我们通过在斑马鱼nbb+/-突变体(Stil基因突变)和野生型斑马鱼中研究发现在对其视网膜注射6-羟多巴(6-OHDA)神经毒素后,视网膜内多巴胺细胞的增殖表现出不同的速率。当Stil基因突变后,多巴胺细胞的增殖减慢,大约只有野生型的一半,而当通过MO-sufu沉默sufu基因变相激活glil基因时,多巴胺细胞的增殖相比野生型要强,而且在损伤后自发的修复过程中,Stil基因与glil基因都参与了细胞增殖过程。说明在斑马鱼视网膜中Stil可以促进多巴胺细胞的增殖。随后,我们以斑马鱼为对象,基于上述成功的实验方法,通过向其眼内注射6-OHDA特异地杀死多巴胺细胞,然后通过行为学和生理学上的鉴定,发现其特征均符合帕金森病人的生理特征,从而建立了一种斑马鱼的帕金森模型。该模型构建简单、快捷,可用于对治疗帕金森氏病药物进行大规模的筛选。
[Abstract]:Cell proliferation is common in nature. From the cell division of lower animals and plants to the growth and development of the advanced organisms, the proliferation of cells is strictly and accurately regulated during the growth and development of the organism. Once the function of a part of the network is regulated, the cell proliferation will be disrupted. And then affect the normal growth and development of organism or the realization of physiological function.
The regeneration and repair of tissues and organs can not be separated from cell proliferation, and the regeneration and repair of tissues and organs are achieved through the continuous proliferation and division of cells. When some tissues or organs of the organism are damaged, the corresponding signal pathways will be started to regulate and realize the repair of tissues and organs. It shows stronger regeneration ability than higher organisms, such as Hydra, vortex, even Xenopus, fish, salamander, and so on, while mammals, as higher organisms, have lost some regenerative ability in long evolution. The zebrafish used in this experiment has a strong regenerative energy, and its genome is also high in the human genome. Homology is of great research significance.
The Stil (SCL/TAL1interrupting locus) gene, which was first found in acute T cell lymphatic leukaemia patients, was found to lead to human congenital small head deformities with the mutation of the gene, and it also regulates the right and left symmetrical development of the mice by the role of the Shh signaling pathway. It is found that the gene is up-regulated in multiple cancer cells. Further studies have found that the expression product of the Stil gene is a component of the centrosome, which plays an important role in the assembly and replication of the centrosome. When the Stil gene is missing, the centrosome will be missing in the cells, and 2 or more centers appear in the cells when the Stil is expressed. Body.
In this experiment, we first studied the PC12 neural cell model, and found that the localization of STIL protein in PC12 cells not only exists in the cytoplasm, but also in the nucleus, suggesting that the Stil gene may have other unknown functions. We also confirmed that the STIL in the PC12 cells can also interact with SUFU and GLI1, and it may be different. In the form of protein complex of the source tripolymer, the cell proliferation slowed down when the Stil gene was silenced, and the expression of the marker gene glil of the Shh signaling pathway was also downregulated. When the Stil gene was overexpressed, the proliferation rate of PC12 cells increased and the expression of glil increased, and the expression of the target gene cyclinD2 was also elevated. At this point, we speculated Stil base. It is possible to regulate the expression of cyclinD2 by regulating the glil gene, and then affect the activity of the cyclin dependent kinase CDK, thus realizing the regulation of the cell cycle.
In addition, we found that the proliferation of dopamine cells in the retina showed different rates after the injection of 6- hydroxydopa (6-OHDA) neurotoxin to the retina of the zebrafish nbb+/- mutant (Stil gene mutation) and wild zebrafish. When the Stil gene mutation, the proliferation of dopamine cells slowed down, about only the wild type. When the glil gene is activated by the MO-sufu silencing of the Sufu gene, the proliferation of the glil gene is stronger than that of the wild type. In the process of spontaneous repair, both the Stil gene and the glil gene are involved in the proliferation of the cells. It shows that Stil can promote the proliferation of dopamine cells in the zebrafish retina.
Then, we used zebrafish as the target, based on the above successful experimental method, to kill dopamine by injecting 6-OHDA into the eye, and then through behavioral and physiological identification, we found that the characteristics of the zebrafish were conformed to the physiological characteristics of Parkinson patients, thus a Parkinson model of zebrafish was established. Single, quick, can be used for large-scale screening of drugs for treating Parkinson's disease.

【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.5

【共引文献】