發(fā)布時間：2018-03-16 19:18

  本文選題：惡性瘧原蟲　切入點：MicroRNAs　出處：《北京協(xié)和醫(yī)學(xué)院》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：瘧疾是經(jīng)按蚊叮咬而感染瘧原蟲所引起的傳染病,嚴(yán)重危害人類健康。能感染人的瘧原蟲有5種,其中惡性瘧原蟲的毒力最強(qiáng),致死率最高。紅內(nèi)期惡性瘧原蟲寄生于人成熟紅細(xì)胞中,這導(dǎo)致它們之間存在錯綜復(fù)雜的相互作用關(guān)系。microRNAs是種廣泛存在于真核生物中的長度-22nt的單鏈非編碼RNA,其在細(xì)胞質(zhì)中與Dicer、Argonaute(AGO)等蛋白形成復(fù)合物而誘導(dǎo)基因沉默,主要通過堿基互補(bǔ)配對與靶基因mRNA的UTR或者CDS結(jié)合,使靶mRNA脫腺苷化、降解和抑制翻譯。最近,LaMonte等人的研究顯示宿主miRNA-451和let-7i移位到紅內(nèi)期惡性瘧原蟲蟲體中,與惡性瘧原蟲PKA-R轉(zhuǎn)錄本發(fā)生嵌合來抑制調(diào)節(jié)性PKA亞基的翻譯,進(jìn)而抑制蟲體的生長；同樣,在鼠中,發(fā)現(xiàn)鼠的miR-155能夠增強(qiáng)小鼠對瘧原蟲的免疫力：在岡比亞按蚊中,岡比亞按蚊的aga-miR-305能夠提高岡比亞按蚊對瘧疾蟲的免疫力。這些研究結(jié)果都表明宿主microRNAs能對瘧原蟲基因進(jìn)行調(diào)控來對抗瘧疾。人miR-185是本實驗室在惡性瘧原蟲3D7里發(fā)現(xiàn)的宿主miRNA之一,且含量很高,通過預(yù)測得知其和var (pfl1955w)的dbl序列靶向互補(bǔ),提示人miR-185可能調(diào)控惡性瘧原蟲var基因和功能。有相關(guān)文獻(xiàn)報道稱,人miR-185在對腫瘤和代謝性疾病的調(diào)控起重要作用,如miR-185負(fù)向調(diào)控雄激素受體抑制前列腺癌細(xì)胞；miR-185靶向原癌基因Sixl抑制腫瘤的生長,靶向SOCS3基因抑制糖尿病功能紊亂等等。為了探討人miR-185對惡性瘧原蟲var (pfl1955w)基因表達(dá)調(diào)控并觀察感染紅細(xì)胞粘附所受到的影響,我們體外培養(yǎng)293T細(xì)胞和惡性瘧原蟲3D7,構(gòu)建雙熒光素酶報告質(zhì)粒和表達(dá)質(zhì)粒,再分別用Lipofectamine 2000轉(zhuǎn)染293T細(xì)胞和Cytomix電轉(zhuǎn)到惡性瘧原蟲3D7,流式細(xì)胞儀檢測轉(zhuǎn)染效率,用雙熒光素酶報告驗證人miR-185對var(pfl1955w)的dbl區(qū)域直接調(diào)控作用,Q-PCR檢測var (pfl1955w) mRNA的表達(dá),細(xì)胞粘附實驗檢測感染紅細(xì)胞的粘附。結(jié)果顯示,轉(zhuǎn)染293T細(xì)胞和惡性瘧原蟲3D7的效率在50%以上；過表達(dá)人miR-185可顯著抑制含var (pfl1955w) dbl的熒光素酶基因的表達(dá)(p0.001)；在293T細(xì)胞和惡性瘧原蟲3D7中,過表達(dá)人miR-185能夠減少·var (pfl1955w)mRNA的含量(p0.05),并使感染紅細(xì)胞粘附作用下降了39%。綜上表明,人miR-185對瘧原蟲var(pfl1955w)基因表達(dá)有下調(diào)作用,進(jìn)而影響惡性瘧原蟲的粘附。
[Abstract]:Malaria is an infectious disease caused by the infection of Plasmodium falciparum through the bite of Anopheles mosquito, which seriously endangers human health. There are five species of Plasmodium falciparum that can infect human beings, among which Plasmodium falciparum is the most virulent. The highest fatality rate. Plasmodium falciparum parasites in human mature red blood cells, This leads to a complex interaction between them. MicroRNAs are single-stranded, -22nt noncoding RNAs that are widespread in eukaryotes and form complexes in the cytoplasm with proteins such as Dicerus ArgonauteAGO.MicroRNAs induce gene silencing. The target mRNA was deadenylized, degraded and inhibited translation by base complementary pairing with UTR or CDS of the target gene mRNA. Recent studies by LaMonte et al showed that the host miRNA-451 and let-7i were translocated into the erythroid parasite of Plasmodium falciparum. Chimerism with Plasmodium falciparum PKA-R transcripts inhibits the translation of regulatory PKA subunits and thus inhibits the growth of parasites. Similarly, in mice, miR-155 has been found to enhance the immunity of mice to Plasmodium: in Anopheles gambiae, The aga-miR-305 of Anopheles gambiae can improve the immunity of Anopheles gambiae against malaria. These results indicate that host microRNAs can regulate the gene of Plasmodium falciparum against malaria. Human miR-185 is distributed in our laboratory in Plasmodium falciparum 3D7. One of the current hosts, miRNA, It was found that the dbl sequence of var pfl1955w was targeted to complement each other, suggesting that human miR-185 might regulate the var gene and function of Plasmodium falciparum. It has been reported that human miR-185 plays an important role in the regulation of tumor and metabolic diseases. For example, the negative regulation of androgen receptor by miR-185 inhibits the growth of prostate cancer cell line miR-185 targeting proto-oncogene Sixl. In order to investigate the effect of human miR-185 on the gene expression of Plasmodium falciparum var pfl1955w, and to observe the effect of human miR-185 on the adhesion of infected erythrocytes, We cultured 293T cells and Plasmodium falciparum 3D7 in vitro, constructed double luciferase reporter plasmids and expressed plasmids, then transfected 293T cells and Cytomix into Plasmodium falciparum 3D7 by Lipofectamine 2000, respectively, and detected the transfection efficiency by flow cytometry. Double luciferase report was used to verify the direct regulation of human miR-185 on the dbl region of varpfl1955). Q-PCR was used to detect the expression of varpfl1955 mRNA, and cell adhesion assay was used to detect the adhesion of infected erythrocytes. The results showed that the efficiency of transfection of 293T cells and Plasmodium falciparum 3D7 was more than 50%. Overexpression of human miR-185 could significantly inhibit the expression of luciferase gene containing var pfl1955w dbl, and in 293T cells and Plasmodium falciparum 3D7, overexpression of human miR-185 could decrease the content of 路var pflfl1955w dbl and decrease the adhesion of infected erythrocytes. Human miR-185 down-regulated the gene expression of Plasmodium falciparum varpfl1955w, and then affected the adhesion of Plasmodium falciparum.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R382

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