發(fā)布時(shí)間：2018-01-24 23:39

  本文關(guān)鍵詞： 瘧疾 惡性瘧原蟲(chóng) RUF6-15 Var　出處：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：瘧疾是一種十分嚴(yán)重的熱帶病,在感染人的4中瘧原蟲(chóng)中惡性瘧原蟲(chóng)的毒性最強(qiáng),死亡率極高。瘧原蟲(chóng)的生活史復(fù)雜,需要兩種類型的宿主-雌性按蚊和人。在人體內(nèi)有紅外期和紅內(nèi)期兩個(gè)發(fā)育階段,但是其主要臨床癥狀發(fā)生在紅內(nèi)期階段。全基因組測(cè)序的完成及后繼的芯片技術(shù)、高通量測(cè)序技術(shù)的快速發(fā)展,惡性瘧原蟲(chóng)中的非編碼RNA (ncRNA)也越來(lái)越引起人們的關(guān)注。ncRNA從長(zhǎng)度上可以分為3類：小于50 nt的小片段ncRNA; 50 nt到500 nt的中等長(zhǎng)度ncRNA;大于500 nt的長(zhǎng)鏈ncRNA (Long non-coding RNA, lncRNA)。通過(guò)數(shù)據(jù)庫(kù)數(shù)據(jù)比較基因組學(xué)分析,在惡性瘧原蟲(chóng)中沒(méi)有找到Dicer、Piwi、PAZ或RdRp的同源區(qū)域,因此認(rèn)為惡性瘧原蟲(chóng)中沒(méi)有內(nèi)源性小RNAs的存在。因此,中等長(zhǎng)度的ncRNA引起了廣泛關(guān)注。對(duì)于瘧原蟲(chóng)中等長(zhǎng)度組成型ncRNA的研究比較成熟,如tRNA、rRNA這些RNA不被翻譯成蛋白質(zhì),但是參與蛋白質(zhì)的翻譯過(guò)程：此外還有snRNA、 snoRNA等參與RNA剪接和RNA修飾。Chakrabarti等鑒定出6個(gè)新的未知功能的RNA (RNAs of unknown function,簡(jiǎn)稱RUFs),其中RUF5、6僅在惡性瘧原蟲(chóng)與里氏瘧原蟲(chóng)中存在,RUF6包括15條高度同源的序列,長(zhǎng)度在144-160 nt,這些同源序列位于RIFIN或VAR的基因間區(qū)。惡性瘧原蟲(chóng)變異抗原基因(Var基因)家族編碼的惡性瘧原蟲(chóng)紅細(xì)胞膜蛋白1(PfEMP1)是介導(dǎo)惡性瘧原蟲(chóng)抗原變異和紅細(xì)胞粘附微血管的媒介。PfEMP1由惡性瘧原蟲(chóng)分泌產(chǎn)生,然后轉(zhuǎn)移到感染的紅細(xì)胞膜上,并在紅細(xì)胞表面蓄積而形成結(jié)節(jié),PfEMP1介導(dǎo)的細(xì)胞粘附和惡性瘧原蟲(chóng)的致病性密切相關(guān)。這些RUFs是否參與了Var基因的調(diào)控以及調(diào)節(jié)瘧原蟲(chóng)紅細(xì)胞內(nèi)期的發(fā)育分化及其內(nèi)在調(diào)節(jié)機(jī)制仍不清楚。本研究通過(guò)Northern blot、FISH、轉(zhuǎn)染、RNA-Seq、qRT-PCR、雙熒光素酶實(shí)驗(yàn)等實(shí)驗(yàn)首次揭示了RUF6中第15條同源簇(RUF6-15)的生物學(xué)功能。在野生型3D7蟲(chóng)株每一個(gè)生活周期中RUF6-15中的轉(zhuǎn)錄水平是隨著生長(zhǎng)發(fā)育時(shí)間的延長(zhǎng)越來(lái)越多,轉(zhuǎn)錄后位于細(xì)胞核內(nèi)參與調(diào)控作用。轉(zhuǎn)染過(guò)表達(dá)RUF6-15的蟲(chóng)株與對(duì)照株相比生長(zhǎng)速度更快,敲低RUF6-15的蟲(chóng)株與對(duì)照株相比生長(zhǎng)速度變慢。通過(guò)過(guò)表達(dá)RUF6-15后轉(zhuǎn)錄本水平的高通量測(cè)序結(jié)果及實(shí)驗(yàn)室驗(yàn)證結(jié)果顯示,RUF6-15的靶基因?yàn)関ar基因家族。進(jìn)一步通過(guò)雙熒光素酶實(shí)驗(yàn)驗(yàn)證了RUF6-15是通過(guò)結(jié)合Var基因5’UTR區(qū)來(lái)調(diào)控var的表達(dá)。粘附實(shí)驗(yàn)表明過(guò)表達(dá)RUF6-15的蟲(chóng)株粘附能力有明顯的提升,RUF6-15敲低的蟲(chóng)株粘附能力有所降低。本研究首次揭示了RUF6-15的生物學(xué)功能及作用機(jī)制,對(duì)深入認(rèn)識(shí)瘧疾的發(fā)病機(jī)制和制定新的防治措施均意義重大。
[Abstract]:Malaria is a very serious tropical disease. The virulence and mortality of Plasmodium falciparum are the highest among the 4 infected people. The life history of Plasmodium falciparum is complex. Two types of hosts are needed-female Anopheles and humans. There are two stages of development in the human body: infrared and red. However, the main clinical symptoms occurred in the red phase. Complete genome sequencing and subsequent chip technology, high-throughput sequencing technology rapid development. The non-coding RNA ncRNAs in Plasmodium falciparum have attracted more and more attention. The length of ncRNAs can be divided into three categories: small fragments of ncRNAs less than 50 NT; Medium length ncRNAs from 50 NT to 500 NT; Long strand ncRNA long non-coding, LNC RNAs > 500nt were used to compare genomics analysis with database data. There is no homologous region of Dicerus Piwipas PAZ or RdRp in Plasmodium falciparum, so it is believed that there is no endogenous small RNAs in Plasmodium falciparum. Medium-length ncRNA has attracted much attention. The studies on the medium-length ncRNA of Plasmodium malaria are more mature, such as tRNA-rRNA, which are not translated into proteins. But involved in the protein translation process: there is also snRNA. SnoRNA et al. participated in RNA splicing and RNA modification. Chakrabarti et al. identified six new unknown functions of RNA (. RNAs of unknown function. Among them, RUF6 contains only 15 highly homologous sequences in Plasmodium falciparum and Plasmodium Rei, with a length of 144-160 NT. These homologous sequences are located in the intergenic region of RIFIN or VAR. Plasmodium falciparum variant antigen gene (Var) family encodes erythrocyte membrane protein 1 of Plasmodium falciparum PfEMP1). PfEMP1 is a vector that mediates the variation of Plasmodium falciparum antigen and the adhesion of erythrocytes to microvessels. PfEMP1 is secreted by Plasmodium falciparum. It then metastasizes to the infected erythrocyte membrane and accumulates on the surface of the erythrocyte to form nodules. The cell adhesion mediated by PfEMP1 is closely related to the pathogenicity of Plasmodium falciparum. Do these RUFs participate in the regulation of Var gene and regulate the development and differentiation of the erythrocyte phase of Plasmodium falciparum and its intrinsic regulation? The mechanism is still unclear. This study is based on Northern. Blot. Fish, transfection of RNA-Seqsil-qRT-PCR. Double luciferase experiments revealed for the first time the 15th homology cluster in RUF6, RUF6-15). The transcriptional level of RUF6-15 in each life cycle of wild-type 3D7 strain is increasing with the prolongation of growth and development time. The post-transcriptional locus involved in the regulation of the cell nucleus. The growth rate of the transfected RUF6-15 strain was faster than that of the control strain. The growth rate of the strain with low RUF6-15 was slower than that of the control strain. The results of high throughput sequencing and laboratory verification showed that the expression of RUF6-15 post-transcripts level was higher than that of the control strain. The target gene of RUF6-15 is the var gene family. Further, the double luciferase experiment proved that RUF6-15 regulates the expression of var by binding to the 5UTR region of Var gene. The results showed that the adhesion ability of RUF6-15 overexpression strains was significantly improved. The adhesion ability of RUF6-15 knockout was decreased. This study revealed the biological function and mechanism of RUF6-15 for the first time. It is of great significance to understand the pathogenesis of malaria and to establish new control measures.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R382

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張小萍;抗惡性瘧原蟲(chóng)再感染的影響因素[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));2001年01期

2 張國(guó)瑩;法屬圭亞那惡性瘧原蟲(chóng)有限的遺傳多樣性和較高的自交率[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));2001年01期

3 陳慧紅;惡性瘧原蟲(chóng)基因組結(jié)構(gòu)研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));2001年04期

4 伍忠鑾,吳忠道,余新炳;惡性瘧原蟲(chóng)基因組的研究進(jìn)展[J];熱帶醫(yī)學(xué)雜志;2004年01期

5 劉德全;;惡性瘧原蟲(chóng)培養(yǎng):建立新株[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));1979年05期

6 何登賢;;惡性瘧原蟲(chóng)體外連續(xù)培養(yǎng)[J];廣西醫(yī)學(xué)院學(xué)報(bào);1979年04期

7 Jensen T.B.TragerW.;王恒禮;;惡性瘧原蟲(chóng)培養(yǎng):另外株的建立[J];河南預(yù)防醫(yī)學(xué)雜志;1979年02期

8 周銘賢;;惡性瘧原蟲(chóng)連續(xù)培養(yǎng)中配子體發(fā)生的觀察[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));1980年02期

9 邱持平;一種連續(xù)培養(yǎng)惡性瘧原蟲(chóng)的精密半自動(dòng)系統(tǒng)[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));1982年01期

10 郭傳坤;;泰國(guó)惡性瘧原蟲(chóng)對(duì)奎寧的敏感性觀察[J];國(guó)外醫(yī)學(xué)(寄生蟲(chóng)病分冊(cè));1984年02期

相關(guān)會(huì)議論文 前10條

1 陳守義;徐勁;李軍濤;武忠鑾;胡旭初;余新炳;;惡性瘧原蟲(chóng)海南株端粒酶催化亞基基因的克隆及序列分析[A];中國(guó)動(dòng)物學(xué)會(huì)原生動(dòng)物學(xué)分會(huì)第十二次學(xué)術(shù)討論會(huì)論文摘要匯編[C];2003年

2 徐勁;葉苓;吳忠道;陳守義;余新炳;;惡性瘧原蟲(chóng)己糖轉(zhuǎn)運(yùn)體編碼基因的初步研究[A];中國(guó)動(dòng)物學(xué)會(huì)第七屆全國(guó)青年寄生蟲(chóng)學(xué)工作者學(xué)術(shù)討論會(huì)論文摘要集[C];2002年

3 雷俊川;繆軍;李s，

本文編號(hào)：1461351