BTV及其基因组dsRNA分子诱导细胞IFN-β和凋亡的比较研究

发布时间：2018-04-25 11:00

本文选题：BTV + dsRNA　；参考：《武汉大学》2005年硕士论文

【摘要】：干扰素(Interferon,IFN)是一类广谱抗病毒物质,是由培养的细胞或人体及动物体因病毒感染或其他干扰素诱生剂(Interferon inducer)作用所产生的一类非特异性抗病毒和抗肿瘤物质。能诱导IFN产生的物质统称为干扰素诱导剂,理想的能直接应用于人体内诱生IFN的IFN诱导剂目前首推多聚肌苷酸(Polyinosinic Polycytidylic Acid,Poly[I:C],简称PIC),但近来发现PIC在人体和其他灵长类体内易被血清核酸酶破坏,诱生IFN的效能较低。据此,国内外科研工作者正在努力寻求和发展新的能用于人体的干扰素诱导剂。本文拟探讨不感染人正常细胞,不引起人任何疾病的动物蓝舌病毒及其基因组dsRNA分子是否具有诱导细胞产生IFN的潜能和诱导肿瘤细胞凋亡的能力。蓝舌病毒(Bluetongue Virus,BTV)是呼肠孤病毒科,环状病毒属成员,是引起绵羊和野生反刍动物蓝舌病的病原体,牛、山羊和鹿则是该病毒的储存宿主。BTV的全套遗传信息由10个基因组片段组成,全基因组由19218个bp组成,总分子量为13× 10~6道尔顿,是迄今为止分子量最大的RNA病毒。蓝舌病毒基因组dsRNA的全套遗传信息的特殊结构为其奠定了作为内源性IFN诱导剂的可能性和无限潜力,当提取到的病毒核酸片段进入细胞内时,因为存在于病毒内衣壳上的依赖RNA的RNA聚合酶被去掉以及BTV基因组dsRNA分子非整倍体进入而使该核酸失去了感染性,但却保持病毒双链RNA诱生IFN的生物学功能,而且将比其病毒颗粒对机体而言更安全有效。本文将蓝舌病毒标准株(BTV-10)的基因组dsRNA通过Trizol提取后,利用脂质体转染技术,转染人宫颈癌Hela细胞和人胚肺原代HEL细胞;以BTV-10分别感染 Hela细胞和HEL细胞;以及直接将裸露的dsRNA加入两种细胞培养物,以比较研究这三种处理后细胞形态学差异,利用检测β-IFN的ELISA试剂盒检测细胞上清液中 IFN含量,并用流式细胞仪检测细胞凋亡率。最终通过统计学方法分析三种处理条件下不同用量的病毒和不同浓度的dsRNA-脂质体复合物及裸露dsRNA产生IFN含量的差异。实验研究发现,BTV及其dsRNA分子均具有诱导细胞产生IFN的生物学功能,
[Abstract]:Interferon Interferon IFN (IFN) is a class of broad-spectrum antiviral substances, consisting of cultured cells or humans and animals. A class of nonspecificity produced by virus infection or other interferon inducer (Interferon inducer). Antiviral and antitumor substances. The substances that can induce the production of IFN are called interferon inducers. The IFN inducer used to induce IFN in human body is currently the first to introduce polyinosinic Polycytidylic acidosis Poly (I: C), but recently it has been found that PIC is susceptible to the nucleation of serum in humans and other primates. The activity of IFN induced by acidase was low. Based on this, researchers at home and abroad are working hard to find and develop new Interferon inducer that can be used in human body. This paper intends to explore the motility that does not infect human normal cells and does not cause any disease in human beings. Does the Bluetongue virus and its Genomic dsRNA molecules have the potential to induce IFN production by cells and to induce tumor fineness The ability to apoptosis. Bluetongue virus (BTV) is a member of the family Reoviridae, a member of the genus Cyclovirus, which causes cotton. The pathogen of blue-tongue disease in sheep and wild ruminants, cattle, goats and deer are the storage hosts of the virus. BTV The nested genetic information consists of 10 genomic fragments and the whole genome consists of 1921 BP with a total molecular weight of 13 脳. One hundred and six Dalton, the largest molecular weight of the RNA virus so far. A complete set of Genomic dsRNA of Bluetongue virus The special structure of transmitting information establishes the possibility and infinite potential as an endogenous IFN inducer. When the viral nucleic acid fragment of the virus enters the cell because of the RNA-dependent RNA polymerase that exists on the virus underwear shell Removal and entry of aneuploidy of BTV genomic dsRNA molecule make the nucleic acid lose its infectivity, but protect The biological function of IFN induced by virus-holding double-stranded RNA will be safer than its viral particles. Effect. The genomic dsRNA of blue tongue virus strain BTV-10 was extracted by Trizol. The plastid transfection technique was used to transfect human cervical cancer Hela cells and human embryonic lung primary HEL cells. Hela cells and HEL cells; and direct addition of bare dsRNA to two cell cultures for comparative study After these three treatments, the cell morphology was different, and the cell supernatant was detected in the supernatant by ELISA kit for detection of 尾 -IFN. IFN content and apoptosis rate were detected by flow cytometry. Finally, the three processing conditions were analyzed by statistical method. Under different doses of virus and different concentrations of dsRNA-liposome complexes and naked dsRNA to produce IFN content Difference. It was found that both BTV and its dsRNA molecules have the biological function of inducing cells to produce IFN.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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