弓形虫棒状体蛋白ROP16导致细胞凋亡和细胞周期阻滞机制及银杏酸杀钉螺药物作用靶向研究

发布时间：2018-07-05 08:41

本文选题：弓形虫 + 蛋白质相互作用　；参考：《复旦大学》2013年硕士论文

【摘要】：第一部分弓形虫棒状体蛋白ROP16导致细胞凋亡和细胞周期阻滞机制研究随着城市的发展和人们生活水平的提高,城市和农村中宠物饲养队伍不断地扩大,同时动物身上可能携带有各种病原微生物,加上忽略饮食卫生等因素,弓形虫感染潜在危险性极度上升。刚地弓形虫(Toxoplasma gondii)是一种专性细胞内寄生原虫,呈世界范围内分布,可感染几乎所有的恒温动物。孕妇感染弓形虫后,通常能将这种感染传给胎儿,如果感染发生在胚胎发育早期可导致脑部疾病、眼病和先天性智障,影响胎儿的发育,导致早产、流产、畸胎和出生缺陷等先天性弓形虫病。调查显示,我国先天性出生缺陷患儿当中弓形虫的感染率达到22%,弱智和精神病患者的发病与弓形虫感染相关。弓形虫入侵人体细胞过程,棒状体蛋白发挥了极其重要的作用。迄今仅发现两种弓形虫棒状体蛋白可侵入人体细胞核内,分别是棒状体蛋白16(ROP16)和PP2C-hn,前者可快速侵入人体细胞核,业已证明ROP16可影响人体细胞的信号转导和转录激活因子(signal transducers and activators of transcription, STAT)信号传导途径从而干扰人体细胞的增殖、分化、凋亡等功能,但该蛋白侵入宿主细胞核后发挥的具体功能目前了解十分有限,需要进一步进行研究。我们的假设是：1、弓形虫棒状体蛋白ROP16可以迅速侵入人体细胞核,就可能会影响到人体细胞的正常生理功能；2、既然弓形虫对脑细胞有倾向性,那么侵入脑细胞的弓形虫就可能影响这些细胞的增殖、分化、凋亡等生理功能,同样ROP16入核后,可影响细胞核功能。3、既然弓形虫感染胚胎脑细胞,那么就有可能影响早期大脑的发育。因此,我们旨在从ROP16功能的基础研究着手,探索ROP16对宿主细胞基因表达谱的影响以及与人体相互作用的核蛋白,获取下游靶基因的信息从中寻找与生长发育相关的因子,以及ROP16与相互作用蛋白结合后对其功能的影响,揭示弓形虫脑病形成以及弓形虫感染导致出生缺陷和智障的机制。本研究首先利用芯片技术检测过表达ROP16对神经细胞基因表达谱的影响,并结合本实验室前期质谱研究,进一步探索ROP16与人体相互作用核蛋白、鉴定及蛋白相互作用,以探讨该蛋白导致宿主细胞凋亡的机制。主要内容及结果如下： (1)通过基因芯片技术检测SH-SY5Y-ROP16稳定转染细胞株表达谱变化,筛选表达差异基因,表达谱芯片分析发现,过表达ROP16的神经母细胞瘤细胞中819基因表达上调,973基因表达下调,差异基因参与多种生物学过程包括神经系统发育,器官形成和增殖凋亡、分化等。 (2)结合前期研究工作质谱的分析结果结果,筛选出与ROP16相互作用的人体核蛋白：以免疫共沉淀双向验证ROP16与p53之间相互结合,经激光共聚焦证实ROP16与p53共定位于宿主细胞核。 (3)ROP16对凋亡相关基因的影响：对上述芯片结果进行验证,在人神经母细胞瘤细胞(SH-SY5Y)中过表达ROP16,采用real-time PCR方法检测凋亡相关基因的表达,发现ROP16可以明显提高p53、BAX、Caspase-9表达,抑制Bcl-2的表达,从而促进细胞凋亡 (4)ROP16与p53相互作用后对其功能的影响：检测过表达ROP16蛋白后对神经细胞凋亡和细胞周期的影响,DNA ladder和流式细胞术检测ROP16对SH-SY5Y细胞凋亡和细胞周期的影响,证明ROP16促进其凋亡,引起细胞周期阻滞,CCK-8实验证实ROP16抑制SH-SY5Y细胞增殖。 (5)ROP16对p53以及不同位点磷酸化的检测：发现ROP16可以促进p53蛋白水平的表达,并提高S20磷酸化水平,降低S37的磷酸化水平。结论：ROP16入侵宿主细胞核后影响宿主细胞基因表达谱。ROP16与p53存在相互作用。ROP16抑制SH-SY5Y细胞的生长。ROP16促进SH-SY5Y细胞的凋亡,引起细胞周期阻滞。弓形虫棒状体蛋白ROP16通过p53蛋白Ser20/37磷酸化引起SH-SY5Y凋亡及细胞周期阻滞。第二部分银杏酸杀钉螺药物作用的靶向研究钉螺(Oncomelania hupensis)属软体动物门(Phylum Mollusca)、腹足纲(Class Gastropoda),.根据形态学上的特征,钉螺属分为两个种：湖北钉螺(Oncomelania hupensis)和微小钉螺(Oncomelania minima)。湖北钉螺国内分布于浙江、江苏,安徽、江西、湖北、湖南、广东及广西；国外尚未发见。钉螺时重要的医学贝类,为日本血吸虫及并殖吸虫的中间宿主。血吸虫病为世界十大热带病之一,全球感染血吸虫病者约为2亿人,近6亿人口受到威胁,有76个国家受影响,其中绝大多数为发展中国家,该疾病已严重危害了这些国家人民的健康和经济发展。日本血吸虫病也是我国最严重的流行病之一,以湖沼型流行区如湖南、湖北、江西、安徽和江苏占全国绝大部分流行面积,全国共有435个血吸虫病流行县(区,市)。虽然我国开展大规模防治工作,使该病流行得到有效控制并取得了非凡的成就,但近年来,由于水灾、人口流动等许多综合因素的影响,血吸虫病开始反弹,疫情局部传播严重,新的疫区增加和有向城市蔓延的迹象表明我国血吸虫病防止任务非常艰巨。因此,对血吸虫病的防治成为我国可持续发展的战略目标之一。在前期研究中,我们已证实银杏酸具有很好的杀螺活性,银杏酸处理钉螺后可以明显抑制其上爬速度,说明银杏酸可作为优良杀螺剂加以研发。鉴于银杏酸可有效抑制钉螺的上爬,使钉螺的活动能力大大下降,表明其供能受阻。其作用机制必然与干扰钉螺的供能有关,从而发挥其杀灭钉螺的作用。本项研究是对前期工作的进一步延续和拓宽,主要通过电镜观察线粒体形态学变化和分子生物学观察基因表达水平变化,来研究银杏酸杀螺机制及其靶点的研究。主要内容及结果如下： (1)分离并纯化银杏酸单体。 (2)银杏酸处理钉螺后明显抑制其上爬率。 (3)电镜证明GA-C13：0显著破坏钉螺线粒体结构,包括嵴断裂溶解、嵴稍肿胀、嵴内腔扩大、空泡形成和膜破裂等；对照组以及国际公认杀螺药物氯硝柳胺对照组均无明显变化。 (4)Real time PCR证实GA-C13:0可以抑制线粒体相关基因的表达,包括细胞色素C氧化酶、细胞色素b、ADH脱氢酶和ATP合酶。结论：钉螺线粒体可能作为银杏酸及其衍生物潜在的药物作用靶点。
[Abstract]:Part one the mechanism of apoptosis and cell cycle arrest induced by Toxoplasma gondii Rodin protein ROP16
With the development of the city and the improvement of people's living standards, pet raising teams in urban and rural areas are constantly expanding, and the potential risk of Toxoplasma infection is extremely rising. Toxoplasma gondii is a special type of intracellular parasite. A worldwide distribution that infects almost all thermostat animals. After infection of Toxoplasma gondii, pregnant women usually transmit this infection to the fetus. If the infection occurs early in the embryonic development, it can lead to brain disease, eye disease, and congenital mental retardation, affecting the development of the fetus, resulting in premature birth, abortion, teratogenesis, and birth defects. The survey showed that the infection rate of Toxoplasma gondii was 22% in children with congenital birth defects in our country, and the incidence of toxoplasmosis was associated with the onset of mentally ill and mentally ill.
The rod like protein plays an extremely important role in the invasion of human cells by Toxoplasma gondii. To date, only two kinds of Toxoplasma protein can invade the nucleus of human body, which are rod protein 16 (ROP16) and PP2C-hn. The former can quickly invade the nucleus of human body. It has been proved that ROP16 can affect the signal transduction and transcription of human cells. The signal transduction pathway of signal transducers and activators of transcription (STAT) interferes with the proliferation, differentiation and apoptosis of human cells. However, the specific functions of this protein after invasion of the host nucleus are very limited and need further study.
Our hypothesis is: 1, the Toxoplasma rod protein ROP16 can quickly invade the human nucleus and may affect the normal physiological function of the human cell. 2, since Toxoplasma gondii is inclined to the brain cells, then the Toxoplasma invading the brain cells may affect the proliferation, differentiation, apoptosis and other physiological functions of these cells, as well as ROP16 entry. Nuclear function can affect the nuclear function.3. Since Toxoplasma infects embryonic brain cells, it may affect the development of early brain. Therefore, we aim to explore the effects of ROP16 on the gene expression profiles of host cells and the nuclear proteins interacting with the human body from the basic research of ROP16 function, and to obtain information from the target genes downstream. Finding factors associated with growth and development, and the effect of the binding of ROP16 and interacting proteins on their function, reveal the formation of Toxoplasma encephalopathy and the mechanism of birth defects and mental retardation caused by Toxoplasma infection.
In this study, we first detected the effect of overexpressed ROP16 on the gene expression profiles of neural cells by using chip technology, and further explored the interaction of ROP16 and human body protein, identification and protein interaction in order to explore the mechanism of apoptosis of the host cell. The main contents and results are as follows:
(1) the expression profiles of SH-SY5Y-ROP16 stable transfected cell lines were detected by gene chip technology, and the differential gene was screened. The expression spectrum chip analysis showed that the 819 gene expression was up regulation in the neuroblastoma cells overexpressing ROP16, the 973 gene expression was down regulated, and the differential gene reference and various biological processes included the development of the nervous system and the organ shape. Apoptosis, differentiation, and so on.
(2) according to the results of the analysis of the previous research work mass spectrometry, the human nuclear proteins interacting with ROP16 were screened: the mutual binding between ROP16 and p53 was verified by the co precipitation of immunoprecipitation, and the confocal laser confocal confirmed that ROP16 and p53 were located in the host nucleus.
(3) the effect of ROP16 on the apoptosis related genes: to verify the results of the above-mentioned chip, overexpress ROP16 in human neuroblastoma cell (SH-SY5Y), and to detect the expression of apoptosis related genes by real-time PCR method. It is found that ROP16 can obviously improve the expression of p53, BAX, Caspase-9, and inhibit the expression of Bcl-2, thus promoting the apoptosis of the cells.
(4) the effect of the interaction of ROP16 and p53 on its function: the effect of ROP16 protein on apoptosis and cell cycle after expression of ROP16 protein, DNA ladder and flow cytometry were used to detect the effect of ROP16 on the apoptosis and cell cycle of SH-SY5Y cells, which proved that ROP16 promoted apoptosis, led to cell cycle arrest, and CCK-8 experiment confirmed ROP16 inhibition SH-SY. 5Y cell proliferation.
(5) detection of phosphorylation of p53 and different loci by ROP16: it is found that ROP16 can promote the expression of p53 protein level, improve the level of S20 phosphorylation and decrease the phosphorylation level of S37.
Conclusion: ROP16 invasion of host cell nucleus affects the interaction of host cell gene expression spectrum.ROP16 and p53,.ROP16 inhibits the growth of SH-SY5Y cell growth.ROP16 to promote apoptosis of SH-SY5Y cells and cause cell cycle arrest. The apoptosis and cell cycle arrest of Toxoplasma gondii ROP16 through p53 protein Ser20/37 phosphorylation.
The second part is targeted research on the effect of ginkgolic acid on snail killing drugs.
The Oncomelania snails (Oncomelania hupensis) belong to the mollusk gate (Phylum Mollusca) and the gastropods (Class Gastropoda). According to the morphological characteristics, the Oncomelania snails are divided into two species: the Hubei snails (Oncomelania hupensis) and the tiny snails (Oncomelania minima). The Hubei nail snails are distributed in Zhejiang, Jiangsu, Anhui, Jiangxi, Hubei, Hunan, Guangdong and Guangzhou. Western; abroad have not yet been seen. The important medical shellfish of Oncomelania are the intermediate host of Schistosoma japonicum and Paragonimus. Schistosomiasis is one of the ten most tropical diseases in the world. About 200 million people infected with schistosomiasis, nearly 600 million of the population are threatened, and 76 countries are affected, most of them are developing countries, and the disease is seriously endangered. The health and economic development of the people of these countries has been harmed. Schistosomiasis is one of the most serious epidemic diseases in China. The epidemic areas of the lake and marshes, such as Hunan, Hubei, Jiangxi, Anhui and Jiangsu, are the most popular areas in the country. There are 435 counties (districts and cities) in China. The epidemic has been effectively controlled and achieved remarkable achievements, but in recent years, due to the influence of flood, population flow and many other comprehensive factors, schistosomiasis has begun to rebound, the local spread of the epidemic is serious, the new epidemic areas are increasing and the spread to the city shows that the prevention of schistosomiasis in China is very difficult. Therefore, the prevention and control of schistosomiasis in China. It has become one of the strategic goals of our country's sustainable development.
In the previous study, we have confirmed that ginkgo acid has good snail activity. Ginkgo acid can obviously inhibit the climbing speed after treatment of Oncomelania Snail. It shows that ginkgo acid can be used as a good snail to develop. The system is bound to interfere with the energy supply of oncomelania, so as to play its role in killing oncomelania snails. This study is a further continuation and widening of early work. The study of the mechanism and target of ginkgo acid snail killing by observing the morphological changes of mitochondria and the changes of gene expression level in molecular biology by electron microscopy. The results are as follows:
(1) isolation and purification of ginkgolic acid monomers.
(2) ginkgolic acid significantly inhibited the climbing rate after treatment of Oncomelania hupensis.
(3) the electron microscope showed that GA-C13:0 significantly destroyed the mitochondria structure of Oncomelania snails, including the lysis of crista fracture, the swelling of the crista slightly, the enlargement of the crista cavity, the formation of vacuoles and the rupture of the membrane, and the control group and the internationally recognized snail drug niclosamide control group had no obvious changes.
(4) Real time PCR confirmed that GA-C13:0 could inhibit the expression of mitochondrial related genes, including cytochrome C oxidase, cytochrome b, ADH dehydrogenase and ATP synthase.
Conclusion: mitochondria from Oncomelania hupensis may be a potential drug target for ginkgolic acids and their derivatives.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R531.8

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