人源miR-185通过靶向恶性疟原虫var基因减弱感染红细胞粘附作用的研究

发布时间：2018-03-16 19:18

本文选题：恶性疟原虫　切入点：MicroRNAs　出处：《北京协和医学院》2016年硕士论文　论文类型：学位论文

【摘要】：疟疾是经按蚊叮咬而感染疟原虫所引起的传染病,严重危害人类健康。能感染人的疟原虫有5种,其中恶性疟原虫的毒力最强,致死率最高。红内期恶性疟原虫寄生于人成熟红细胞中,这导致它们之间存在错综复杂的相互作用关系。microRNAs是种广泛存在于真核生物中的长度-22nt的单链非编码RNA,其在细胞质中与Dicer、Argonaute(AGO)等蛋白形成复合物而诱导基因沉默,主要通过碱基互补配对与靶基因mRNA的UTR或者CDS结合,使靶mRNA脱腺苷化、降解和抑制翻译。最近,LaMonte等人的研究显示宿主miRNA-451和let-7i移位到红内期恶性疟原虫虫体中,与恶性疟原虫PKA-R转录本发生嵌合来抑制调节性PKA亚基的翻译,进而抑制虫体的生长；同样,在鼠中,发现鼠的miR-155能够增强小鼠对疟原虫的免疫力：在冈比亚按蚊中,冈比亚按蚊的aga-miR-305能够提高冈比亚按蚊对疟疾虫的免疫力。这些研究结果都表明宿主microRNAs能对疟原虫基因进行调控来对抗疟疾。人miR-185是本实验室在恶性疟原虫3D7里发现的宿主miRNA之一,且含量很高,通过预测得知其和var (pfl1955w)的dbl序列靶向互补,提示人miR-185可能调控恶性疟原虫var基因和功能。有相关文献报道称,人miR-185在对肿瘤和代谢性疾病的调控起重要作用,如miR-185负向调控雄激素受体抑制前列腺癌细胞；miR-185靶向原癌基因Sixl抑制肿瘤的生长,靶向SOCS3基因抑制糖尿病功能紊乱等等。为了探讨人miR-185对恶性疟原虫var (pfl1955w)基因表达调控并观察感染红细胞粘附所受到的影响,我们体外培养293T细胞和恶性疟原虫3D7,构建双荧光素酶报告质粒和表达质粒,再分别用Lipofectamine 2000转染293T细胞和Cytomix电转到恶性疟原虫3D7,流式细胞仪检测转染效率,用双荧光素酶报告验证人miR-185对var(pfl1955w)的dbl区域直接调控作用,Q-PCR检测var (pfl1955w) mRNA的表达,细胞粘附实验检测感染红细胞的粘附。结果显示,转染293T细胞和恶性疟原虫3D7的效率在50%以上；过表达人miR-185可显著抑制含var (pfl1955w) dbl的荧光素酶基因的表达(p0.001)；在293T细胞和恶性疟原虫3D7中,过表达人miR-185能够减少·var (pfl1955w)mRNA的含量(p0.05),并使感染红细胞粘附作用下降了39%。综上表明,人miR-185对疟原虫var(pfl1955w)基因表达有下调作用,进而影响恶性疟原虫的粘附。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R382

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