本文選題：弓形蟲(chóng) + 蛋白質(zhì)相互作用　； 參考：《復(fù)旦大學(xué)》2013年碩士論文

【摘要】：第一部分弓形蟲(chóng)棒狀體蛋白R(shí)OP16導(dǎo)致細(xì)胞凋亡和細(xì)胞周期阻滯機(jī)制研究 隨著城市的發(fā)展和人們生活水平的提高,城市和農(nóng)村中寵物飼養(yǎng)隊(duì)伍不斷地?cái)U(kuò)大,同時(shí)動(dòng)物身上可能攜帶有各種病原微生物,加上忽略飲食衛(wèi)生等因素,弓形蟲(chóng)感染潛在危險(xiǎn)性極度上升。剛地弓形蟲(chóng)(Toxoplasma gondii)是一種專(zhuān)性細(xì)胞內(nèi)寄生原蟲(chóng),呈世界范圍內(nèi)分布,可感染幾乎所有的恒溫動(dòng)物。孕婦感染弓形蟲(chóng)后,通常能將這種感染傳給胎兒,如果感染發(fā)生在胚胎發(fā)育早期可導(dǎo)致腦部疾病、眼病和先天性智障,影響胎兒的發(fā)育,導(dǎo)致早產(chǎn)、流產(chǎn)、畸胎和出生缺陷等先天性弓形蟲(chóng)病。調(diào)查顯示,我國(guó)先天性出生缺陷患兒當(dāng)中弓形蟲(chóng)的感染率達(dá)到22%,弱智和精神病患者的發(fā)病與弓形蟲(chóng)感染相關(guān)。 弓形蟲(chóng)入侵人體細(xì)胞過(guò)程,棒狀體蛋白發(fā)揮了極其重要的作用。迄今僅發(fā)現(xiàn)兩種弓形蟲(chóng)棒狀體蛋白可侵入人體細(xì)胞核內(nèi),分別是棒狀體蛋白16(ROP16)和PP2C-hn,前者可快速侵入人體細(xì)胞核,業(yè)已證明ROP16可影響人體細(xì)胞的信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子(signal transducers and activators of transcription, STAT)信號(hào)傳導(dǎo)途徑從而干擾人體細(xì)胞的增殖、分化、凋亡等功能,但該蛋白侵入宿主細(xì)胞核后發(fā)揮的具體功能目前了解十分有限,需要進(jìn)一步進(jìn)行研究。 我們的假設(shè)是：1、弓形蟲(chóng)棒狀體蛋白R(shí)OP16可以迅速侵入人體細(xì)胞核,就可能會(huì)影響到人體細(xì)胞的正常生理功能；2、既然弓形蟲(chóng)對(duì)腦細(xì)胞有傾向性,那么侵入腦細(xì)胞的弓形蟲(chóng)就可能影響這些細(xì)胞的增殖、分化、凋亡等生理功能,同樣ROP16入核后,可影響細(xì)胞核功能。3、既然弓形蟲(chóng)感染胚胎腦細(xì)胞,那么就有可能影響早期大腦的發(fā)育。因此,我們旨在從ROP16功能的基礎(chǔ)研究著手,探索ROP16對(duì)宿主細(xì)胞基因表達(dá)譜的影響以及與人體相互作用的核蛋白,獲取下游靶基因的信息從中尋找與生長(zhǎng)發(fā)育相關(guān)的因子,以及ROP16與相互作用蛋白結(jié)合后對(duì)其功能的影響,揭示弓形蟲(chóng)腦病形成以及弓形蟲(chóng)感染導(dǎo)致出生缺陷和智障的機(jī)制。 本研究首先利用芯片技術(shù)檢測(cè)過(guò)表達(dá)ROP16對(duì)神經(jīng)細(xì)胞基因表達(dá)譜的影響,并結(jié)合本實(shí)驗(yàn)室前期質(zhì)譜研究,進(jìn)一步探索ROP16與人體相互作用核蛋白、鑒定及蛋白相互作用,以探討該蛋白導(dǎo)致宿主細(xì)胞凋亡的機(jī)制。主要內(nèi)容及結(jié)果如下： (1)通過(guò)基因芯片技術(shù)檢測(cè)SH-SY5Y-ROP16穩(wěn)定轉(zhuǎn)染細(xì)胞株表達(dá)譜變化,篩選表達(dá)差異基因,表達(dá)譜芯片分析發(fā)現(xiàn),過(guò)表達(dá)ROP16的神經(jīng)母細(xì)胞瘤細(xì)胞中819基因表達(dá)上調(diào),973基因表達(dá)下調(diào),差異基因參與多種生物學(xué)過(guò)程包括神經(jīng)系統(tǒng)發(fā)育,器官形成和增殖凋亡、分化等。 (2)結(jié)合前期研究工作質(zhì)譜的分析結(jié)果結(jié)果,篩選出與ROP16相互作用的人體核蛋白：以免疫共沉淀雙向驗(yàn)證ROP16與p53之間相互結(jié)合,經(jīng)激光共聚焦證實(shí)ROP16與p53共定位于宿主細(xì)胞核。 (3)ROP16對(duì)凋亡相關(guān)基因的影響：對(duì)上述芯片結(jié)果進(jìn)行驗(yàn)證,在人神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y)中過(guò)表達(dá)ROP16,采用real-time PCR方法檢測(cè)凋亡相關(guān)基因的表達(dá),發(fā)現(xiàn)ROP16可以明顯提高p53、BAX、Caspase-9表達(dá),抑制Bcl-2的表達(dá),從而促進(jìn)細(xì)胞凋亡 (4)ROP16與p53相互作用后對(duì)其功能的影響：檢測(cè)過(guò)表達(dá)ROP16蛋白后對(duì)神經(jīng)細(xì)胞凋亡和細(xì)胞周期的影響,DNA ladder和流式細(xì)胞術(shù)檢測(cè)ROP16對(duì)SH-SY5Y細(xì)胞凋亡和細(xì)胞周期的影響,證明ROP16促進(jìn)其凋亡,引起細(xì)胞周期阻滯,CCK-8實(shí)驗(yàn)證實(shí)ROP16抑制SH-SY5Y細(xì)胞增殖。 (5)ROP16對(duì)p53以及不同位點(diǎn)磷酸化的檢測(cè)：發(fā)現(xiàn)ROP16可以促進(jìn)p53蛋白水平的表達(dá),并提高S20磷酸化水平,降低S37的磷酸化水平。 結(jié)論：ROP16入侵宿主細(xì)胞核后影響宿主細(xì)胞基因表達(dá)譜。ROP16與p53存在相互作用。ROP16抑制SH-SY5Y細(xì)胞的生長(zhǎng)。ROP16促進(jìn)SH-SY5Y細(xì)胞的凋亡,引起細(xì)胞周期阻滯。弓形蟲(chóng)棒狀體蛋白R(shí)OP16通過(guò)p53蛋白Ser20/37磷酸化引起SH-SY5Y凋亡及細(xì)胞周期阻滯。 第二部分銀杏酸殺釘螺藥物作用的靶向研究 釘螺(Oncomelania hupensis)屬軟體動(dòng)物門(mén)(Phylum Mollusca)、腹足綱(Class Gastropoda),.根據(jù)形態(tài)學(xué)上的特征,釘螺屬分為兩個(gè)種：湖北釘螺(Oncomelania hupensis)和微小釘螺(Oncomelania minima)。湖北釘螺國(guó)內(nèi)分布于浙江、江蘇,安徽、江西、湖北、湖南、廣東及廣西；國(guó)外尚未發(fā)見(jiàn)。釘螺時(shí)重要的醫(yī)學(xué)貝類(lèi),為日本血吸蟲(chóng)及并殖吸蟲(chóng)的中間宿主。血吸蟲(chóng)病為世界十大熱帶病之一,全球感染血吸蟲(chóng)病者約為2億人,近6億人口受到威脅,有76個(gè)國(guó)家受影響,其中絕大多數(shù)為發(fā)展中國(guó)家,該疾病已嚴(yán)重危害了這些國(guó)家人民的健康和經(jīng)濟(jì)發(fā)展。日本血吸蟲(chóng)病也是我國(guó)最嚴(yán)重的流行病之一,以湖沼型流行區(qū)如湖南、湖北、江西、安徽和江蘇占全國(guó)絕大部分流行面積,全國(guó)共有435個(gè)血吸蟲(chóng)病流行縣(區(qū),市)。雖然我國(guó)開(kāi)展大規(guī)模防治工作,使該病流行得到有效控制并取得了非凡的成就,但近年來(lái),由于水災(zāi)、人口流動(dòng)等許多綜合因素的影響,血吸蟲(chóng)病開(kāi)始反彈,疫情局部傳播嚴(yán)重,新的疫區(qū)增加和有向城市蔓延的跡象表明我國(guó)血吸蟲(chóng)病防止任務(wù)非常艱巨。因此,對(duì)血吸蟲(chóng)病的防治成為我國(guó)可持續(xù)發(fā)展的戰(zhàn)略目標(biāo)之一。 在前期研究中,我們已證實(shí)銀杏酸具有很好的殺螺活性,銀杏酸處理釘螺后可以明顯抑制其上爬速度,說(shuō)明銀杏酸可作為優(yōu)良?xì)⒙輨┘右匝邪l(fā)。鑒于銀杏酸可有效抑制釘螺的上爬,使釘螺的活動(dòng)能力大大下降,表明其供能受阻。其作用機(jī)制必然與干擾釘螺的供能有關(guān),從而發(fā)揮其殺滅釘螺的作用。本項(xiàng)研究是對(duì)前期工作的進(jìn)一步延續(xù)和拓寬,主要通過(guò)電鏡觀(guān)察線(xiàn)粒體形態(tài)學(xué)變化和分子生物學(xué)觀(guān)察基因表達(dá)水平變化,來(lái)研究銀杏酸殺螺機(jī)制及其靶點(diǎn)的研究。主要內(nèi)容及結(jié)果如下： (1)分離并純化銀杏酸單體。 (2)銀杏酸處理釘螺后明顯抑制其上爬率。 (3)電鏡證明GA-C13：0顯著破壞釘螺線(xiàn)粒體結(jié)構(gòu),包括嵴斷裂溶解、嵴稍腫脹、嵴內(nèi)腔擴(kuò)大、空泡形成和膜破裂等；對(duì)照組以及國(guó)際公認(rèn)殺螺藥物氯硝柳胺對(duì)照組均無(wú)明顯變化。 (4)Real time PCR證實(shí)GA-C13:0可以抑制線(xiàn)粒體相關(guān)基因的表達(dá),包括細(xì)胞色素C氧化酶、細(xì)胞色素b、ADH脫氫酶和ATP合酶。 結(jié)論：釘螺線(xiàn)粒體可能作為銀杏酸及其衍生物潛在的藥物作用靶點(diǎn)。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R531.8

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 彭鴻娟;弓形蟲(chóng)棒狀體蛋白研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));1999年05期

2 王惠玲,李秋英,王高華,舒暢,王曉萍,劉浩,段金蓮,楊相升;精神分裂癥首次發(fā)病患者弓形蟲(chóng)感染率及其對(duì)臨床癥狀的影響[J];中華精神科雜志;2005年03期

3 李文姝,陸惠民,閔太善,黃偉達(dá);弓形蟲(chóng)棒狀體蛋白R(shí)OP2基因的克隆與表達(dá)[J];中國(guó)人獸共患病雜志;2005年10期

，

本文編號(hào)：2099634