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

【摘要】：瘧疾是一種十分嚴重的熱帶病,在感染人的4中瘧原蟲中惡性瘧原蟲的毒性最強,死亡率極高。瘧原蟲的生活史復雜,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。全基因組測序的完成及后繼的芯片技術(shù)、高通量測序技術(shù)的快速發(fā)展,惡性瘧原蟲中的非編碼RNA (ncRNA)也越來越引起人們的關(guān)注。ncRNA從長度上可以分為3類：小于50 nt的小片段ncRNA; 50 nt到500 nt的中等長度ncRNA;大于500 nt的長鏈ncRNA (Long non-coding RNA, lncRNA)。通過數(shù)據(jù)庫數(shù)據(jù)比較基因組學分析,在惡性瘧原蟲中沒有找到Dicer、Piwi、PAZ或RdRp的同源區(qū)域,因此認為惡性瘧原蟲中沒有內(nèi)源性小RNAs的存在。因此,中等長度的ncRNA引起了廣泛關(guān)注。對于瘧原蟲中等長度組成型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)的翻譯過程：此外還有snRNA、 snoRNA等參與RNA剪接和RNA修飾。Chakrabarti等鑒定出6個新的未知功能的RNA (RNAs of unknown function,簡稱RUFs),其中RUF5、6僅在惡性瘧原蟲與里氏瘧原蟲中存在,RUF6包括15條高度同源的序列,長度在144-160 nt,這些同源序列位于RIFIN或VAR的基因間區(qū)。惡性瘧原蟲變異抗原基因(Var基因)家族編碼的惡性瘧原蟲紅細胞膜蛋白1(PfEMP1)是介導惡性瘧原蟲抗原變異和紅細胞粘附微血管的媒介。PfEMP1由惡性瘧原蟲分泌產(chǎn)生,然后轉(zhuǎn)移到感染的紅細胞膜上,并在紅細胞表面蓄積而形成結(jié)節(jié),PfEMP1介導的細胞粘附和惡性瘧原蟲的致病性密切相關(guān)。這些RUFs是否參與了Var基因的調(diào)控以及調(diào)節(jié)瘧原蟲紅細胞內(nèi)期的發(fā)育分化及其內(nèi)在調(diào)節(jié)機制仍不清楚。本研究通過Northern blot、FISH、轉(zhuǎn)染、RNA-Seq、qRT-PCR、雙熒光素酶實驗等實驗首次揭示了RUF6中第15條同源簇(RUF6-15)的生物學功能。在野生型3D7蟲株每一個生活周期中RUF6-15中的轉(zhuǎn)錄水平是隨著生長發(fā)育時間的延長越來越多,轉(zhuǎn)錄后位于細胞核內(nèi)參與調(diào)控作用。轉(zhuǎn)染過表達RUF6-15的蟲株與對照株相比生長速度更快,敲低RUF6-15的蟲株與對照株相比生長速度變慢。通過過表達RUF6-15后轉(zhuǎn)錄本水平的高通量測序結(jié)果及實驗室驗證結(jié)果顯示,RUF6-15的靶基因為var基因家族。進一步通過雙熒光素酶實驗驗證了RUF6-15是通過結(jié)合Var基因5’UTR區(qū)來調(diào)控var的表達。粘附實驗表明過表達RUF6-15的蟲株粘附能力有明顯的提升,RUF6-15敲低的蟲株粘附能力有所降低。本研究首次揭示了RUF6-15的生物學功能及作用機制,對深入認識瘧疾的發(fā)病機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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R382

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