装载不同功能RNA的重组MS2病毒样颗粒的原核表达及应用研究

发布时间：2018-06-11 20:53

本文选题：埃博拉病毒 + 室间质量评价　；参考：《北京协和医学院》2016年博士论文

【摘要】：2014年西非爆发的埃博拉出血热疫情在全世界范围引起广泛关注。中国与西非往来密切,疫情爆发期间,埃博拉病患自境外输入中国的风险较高且持续存在。提高该病毒的早期准确检测能力,成为我国防控埃博拉疫情的关键。为提高全国范围,特别是重要港口城市,具备埃博拉病毒检测资质的实验室、检验检疫机构等对该病毒的检测能力,2014年12月卫生部临床检验中心组织开展相关质量评价工作。鉴于埃博拉病毒属于生物安全四级防控病毒,一旦感染具有极高的致死性,常规利用灭活病毒或感染病人阳性血清制备质控品的方法不适用于本次质量评价。因此,我们选择生物安全性高、稳定性好、对RNA保护作用强的噬菌体MS2病毒样颗粒(Bacteriophage MS2 virus-like particles, MS2 VLPs),制备包裹2014年西非流行株扎伊尔型埃博拉病毒核衣壳蛋白(Nucleoprotein, NP) RNA全长、包膜糖蛋白(Glycoprotein, GP)及RNA依赖的RNA聚合酶L(Polymerase)部分RNA序列的重组MS2 VLPs质控品,对各参评机构进行发放。在19家参评机构回报的20份结果中(其中一家机构回报了NP及GP/L双靶标检测结果),14家机构(73.68%)回报完全正确,3家机构(15.79%)出现一个假阴性结果,1家(5.26%)出现两个假阴性结果,剩余1家机构(5.26%)回报错误结果超过3个。本次质评样本盘中全部阴性样本结果回报正确,上述错误回报结果主要集中在对弱阳性样本的检出方面。针对本次质量评价中存在的假阴性问题,我们提出使用商品化试剂盒或国家CDC提供的标准引物、探针检测,使用双靶标检测方法,以及对操作人员规范化培训、及时校准仪器等改进意见。本次质评工作促进了各参评机构埃博拉病毒核酸检测能力的提高,在疫情爆发阶段有效防控了埃博拉病毒流入中国。在第二部分中,为解决之前基于MS2 VLP的miRNA递送体系中,化学交联方法存在的成功率低、成本高、周期长、步骤繁琐等问题,我们采用噬菌体表面展示技术,制备了表面展示细胞穿透肽TAT内含pre-miR122的重组MS2 VLPs用于肝癌治疗研究。我们首先明确上述展示TAT的VLPs是否具备细胞膜穿透功能及递送miR122的能力。为进一步检测内含pre-miR122表面展示TAT的MS2 VLPs对肝细胞癌的抑制效果,我们同时制备了内含pre-miR122表面化学交联TAT的MS2 VLPs做为对比。通过CCK8、transwell小室、流式细胞术、Western blot及CT影像学分析、HE染色、免疫组化等方法,在细胞及动物水平明确上述两种VLPs对肝癌的抑制效果及是否存在差异。结果证明,内含pre-miR122表面展示TAT的MS2 VLPs可成功穿透细胞膜,实现miR122的高效胞内递送。应用表面展示TAT及化学交联TAT的VLPs处理Hep3B、HepG2及Huh7三种肝癌细胞后可发现,上述两种内含pre-miR122的VLPs对三种细胞均存在抑制作用,且表面展示TAT的VLPs抑制效果更加明显。继而我们选择Hep3B细胞(VLPs处理后细胞水平肿瘤抑制效果最明显)建立肝癌动物模型。对荷瘤组小鼠尾静脉注射上述两种VLPs后发现,表面展示TAT及化学交联TAT内含pre-miR122的VLPs均可有效抑制肿瘤生长,且以前者抑制效果更明显。以上结果提示,我们制备的基于表面展示TAT的重组MS2 VLP miR122递送载体具有细胞穿透功能,体内、体外均对肝细胞癌具有抑制作用。与化学交联TAT内含pre-miR122的VLPs相比,展示TAT的VLPs制备过程简单、周期短、肿瘤抑制效果更加显著,展现出良好的应用前景。
[Abstract]:The outbreak of Ebola haemorrhagic fever in West Africa in 2014 has attracted wide attention all over the world. China is close to West Africa. During the outbreak of the outbreak, the risk of Ebola disease from abroad is high and persisted. Improving the early accurate detection ability of the virus has become the key to our national defense control Ebola epidemic. Scope, especially important port cities, laboratories with aptitude test for Ebola virus detection, inspection and quarantine agencies, and so on. In December 2014, the Department of clinical inspection of the Ministry of health organized the relevant quality evaluation work. In view of the four level of the Ebola virus is a biological safety control virus, once the infection has a high death rate. Sex, the routine use of inactivated virus or the positive serum of infected patients to prepare quality control products is not suitable for this quality evaluation. Therefore, we choose the phage MS2 virus like Bacteriophage MS2 virus-like particles (MS2 VLPs), which has high biological safety and good protective effect, to prepare the 2014 West African epidemic strain. The Zaire type Ebola virus nucleocapsid protein (Nucleoprotein, NP) RNA full length, Glycoprotein (GP) and RNA dependent RNA polymerase L (Polymerase) RNA sequence of RNA polymerase L (Polymerase) RNA sequence are restructured MS2 VLPs quality control products. Double target test results), 14 institutions (73.68%) were fully correct, 3 institutions (15.79%) had a false negative result, 1 (5.26%) had two false negative results, and the remaining 1 institutions (5.26%) had more than 3 return error results. The results of all negative samples in this review sample were correct, and the above error returns were mainly concentrated on the results. For the detection of weak positive samples, in view of the false negative problems in this quality evaluation, we put forward some suggestions on the use of commercialized kits or standard primers provided by national CDC, probe detection, the use of double target detection methods, the standardized training of operators, and timely calibration of the instrument. In the second part, in the second part of the miRNA delivery system based on VLP, the success rate of the chemical crosslinking method is low, the cost is high, the cycle is long, and the steps are tedious. We use the phage surface. The recombinant MS2 VLPs containing pre-miR122 in surface display cell penetrating peptide TAT was prepared for the treatment of liver cancer. We first clearly demonstrated that the VLPs of TAT has the ability of cell membrane penetration and delivery of miR122. To further detect the inhibitory effect of MS2 VLPs on pre-miR122 containing TAT, the inhibitory effect of MS2 VLPs on hepatocellular carcinoma. We also made a comparison of MS2 VLPs containing pre-miR122 surface chemically linked TAT. Through CCK8, Transwell chamber, flow cytometry, Western blot and CT imaging analysis, HE staining, immunohistochemistry and other methods, the inhibitory effects and differences of these two VLPs on liver cancer were identified at cell and animal levels. The MS2 VLPs containing TAT on the surface of pre-miR122 can successfully penetrate the cell membrane and achieve the efficient intracellular delivery of miR122. After the application of the VLPs to treat Hep3B, HepG2 and Huh7 three hepatoma cells with the VLPs on the surface of TAT and chemical crosslinked TAT, the above two kinds of pre-miR122 are inhibited on the three cells and are displayed on the surface. The effect was more obvious. Then we selected Hep3B cells (the most obvious inhibitory effect of cell level after VLPs treatment) to establish the animal model of liver cancer. After injection of the two kinds of VLPs in the tail vein of the tumor bearing mice, it was found that the surface display of TAT and the VLPs containing pre-miR122 in the chemically crosslinked TAT could effectively inhibit the growth of the tumor, and the previous inhibition effect was effective. The results suggested that the above results suggest that the recombinant MS2 VLP miR122 delivery carrier based on the surface display TAT has cell penetration, both in vivo and in vitro have inhibitory effect on hepatocellular carcinoma. Compared with the VLPs containing pre-miR122 in chemical crosslinked TAT, the VLPs preparation process of TAT is simple, the cycle is short, and the tumor inhibition effect is more obvious. It shows a good prospect of application.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.8

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