HCV小鼠模型的建立及其在肝脏免疫损伤研究中的应用

发布时间：2018-07-09 21:21

本文选题：丙型病毒性肝炎 + 动物模型　；参考：《中国人民解放军军事医学科学院》2014年博士论文

【摘要】：丙型肝炎病毒(Hepatitis C Virus, HCV)是黄病毒属的单股正链RNA病毒,是慢性肝炎的主要致病因子之一。临床资料显示,丙肝患者的肝细胞极易发生损伤,且肝细胞的自身修复能力减弱、增生异常,最终引发肝纤维化、肝硬化及肝癌的发生,迄今仍缺乏有效的防治手段。加强丙型病毒性肝炎的相关基础研究,是当前丙型病毒性肝炎防治研究中的重要命题。 HCV是一种非细胞毒性的嗜肝病毒,丙肝患者肝细胞极易发生损伤的现象主要是由宿主免疫系统与受病毒感染的肝细胞间的相互作用引起。特别是宿主肝脏免疫系统对被感染肝实质细胞的异常免疫反应导致了肝细胞损伤与修复的平衡失调。但HCV和宿主肝脏免疫系统间相互作用的机制尚不明确,探索HCV和宿主肝脏免疫系统之间的相互作用是本研究的重要目的。本研究分以下两个部分展开： 1.肝脏特异性稳定转染技术的探索与应用合适的动物模型是探索HCV免疫致病机理的重要环节。目前,可感染HCV的动物模型仅限于黑猩猩、嵌合人类肝细胞小鼠模型及多种HCV受体联合转基因小鼠模型。但由于受数量有限、价格昂贵等因素的影响,限制了这几类动物模型在基础研究中的广泛应用。传统的HCV转基因小鼠模型为研究HCV蛋白与宿主之间的相互作用奠定了良好的基础,但由于存在先天免疫耐受,使其不能应用于HCV和肝脏免疫系统间的相互作用研究。基于此,采用合适的方法建立免疫功能正常并适于实验室广泛应用的HCV动物模型是目前抗HCV相关研究的重要环节。 1.1眼底静脉丛水动力转染技术的建立基于经典的水动力转染技术和眼底静脉丛注射技术建立的眼底静脉丛水动力转染技术具有良好的肝脏靶向性,在目前缺乏合适的HCV小动物模型的前提下,通过该方法建立HCV小鼠模型替代HCV体内感染模型可能是一种快速、高效、稳定、简便的构建HCV肝脏特异性转基因动物模型的新方法。传统的水动力转染技术有其自身难以克服的缺点,因此,本研究结合眼底静脉丛输注建立了眼底静脉丛水动力转染技术。该技术是将大量含有目的基因表达质粒的盐水溶液从眼底静脉快速注入实验动物体内,实现外源基因在肝脏特异、高效表达。通过条件摸索,确立了应用该技术的最适转染条件,并分析了其对实验动物器官机能的影响。在转染效率方面,眼底静脉丛水动力转染技术与经典的尾静脉水动力转染技术相当。该技术操作更简单、可重复性强。最为关键的是,眼底静脉丛水动力转染技术克服了尾静脉水动力转染仅能用于尾静脉适合注射的实验动物的应用限制。该技术除可用于常规实验动物外,还可用于尾静脉无法注射或无尾静脉的实验动物,有效拓展了水动力转染技术的应用范围。 1.2密码子优化的噬菌体整合酶介导外源基因在小鼠肝脏长期稳定表达噬菌体整合酶技术是一种可以介导外源基因在靶细胞发生特异性整合的生物酶技术。新近报道的鼠密码子优化的噬菌体整合酶针对鼠类基因特点进行密码子优化,对于鼠源细胞具有更高的整合效率。为促进水动力转染技术导入肝组织中的外源基因在小鼠肝细胞中长期稳定表达,我们在实验中引入鼠密码子优化的噬菌体整合酶技术。并以NF-κB sensor质粒作为报告基因,通过与噬菌体整合酶表达质粒共转染实现对活体小鼠肝脏长期稳定转染的技术优化,建立了可长期监测肝脏NF-κB活性的小动物模型。研究结果显示,与传统的噬菌体整合酶系统相比,鼠密码子优化的噬菌体整合酶可以更加有效的促使外源基因在活体小鼠肝细胞中发生整合,并介导外源基因长期高效表达。 1.3基于水动力转染和整合酶技术建立HCV基因组肝脏长期表达小鼠模型在前期研究工作基础上,我们联合水动力转染技术和鼠密码子优化的噬菌体整合酶技术,基于免疫功能正常的C57BL/6小鼠,建立了HCV全基因组肝脏长期稳定表达的动物模型。此外,在HCV全基因组表达载体上融合了报告基因—萤火虫荧光素酶,HCV在小鼠肝脏的表达情况可以通过活体成像技术进行实时监测,为后续实验提供了方便。 2.HCV促进小鼠肝脏免疫损伤的机制研究强势的肝脏免疫系统是机体抵御外来侵袭的重要防御机制。占肝脏淋巴细胞总数30%左右的NK细胞,对抵抗病毒、寄生菌感染和恶性转化细胞侵润等侵害具有十分重要的意义。但NK细胞是否参与丙肝患者肝细胞损伤的免疫过程尚不明确。因此本研究寻找以NK细胞为代表的肝脏免疫细胞在丙肝患者肝细胞损伤中的作用机制,可能是防治HCV所导致的肝脏疾病的重要方法。 2.1HCV对小鼠肝脏免疫细胞的影响以本研究建立的免疫功能正常的HCV全基因组肝脏长期表达小鼠模型为基础,首先分析HCV表达对肝脏免疫细胞的影响,结果显示：HCV降低了小鼠肝脏NK细胞的数量,但不影响其活性；HCV对小鼠其他类型的肝脏淋巴细胞(NKT、T、DC等)未造成显著影响。这一结果与临床报道相似。 2.2HCV促进Con A诱导的小鼠肝脏损伤及其免疫学机制鉴于NK细胞在肝脏天然免疫系统中发挥的重要作用,我们认为,肝脏NK细胞的减少可能会对机体的免疫反应产生一定的影响。为进一步探索HCV与宿主肝脏免疫系统的关系,基于该HCV小鼠模型,并结合Con A诱导的自身免疫性肝损伤模型,对HCV免疫致病机理进行探索。结果显示：中等剂量Con A刺激后,HCV组小鼠的肝脏免疫损伤现象增强；而高剂量Con A刺激后,HCV小鼠发生严重的自身免疫性肝炎并促使小鼠死亡率增高。进一步研究结果显示：ConA静脉注射诱发小鼠自身免疫性肝炎后,小鼠肝脏免疫细胞活化,其中肝脏天然免疫细胞---NK细胞的数量和功能在HCV组和对照组小鼠间的差异最为明显。选择性清除小鼠体内的NK细胞,则HCV小鼠对Con A诱导的肝损伤的敏感性降低,肝损伤减轻。说明HCV小鼠对Con A诱导的肝损伤的敏感性增强与小鼠肝脏NK细胞的过度活化相关。针对NK细胞功能的研究结果显示,肝脏NK细胞免疫功能过度激活依赖于NK细胞表面激活性受体NKG2D及其胞内与其免疫活性密切相关的细胞因子(IFN-γ、TNF-α等)的表达。同时,NKG2D在肝细胞表面的配体NKG2DL(H60)的表达水平也显著上调。综上所述,本研究结合眼底静脉丛水动力转染技术、密码子优化的噬菌体整合酶和活体荧光成像技术,基于免疫功能正常的C57BL/6小鼠,建立了可视化的HCV肝脏特异性长期表达小鼠模型。研究显示,该模型对Con A诱导的肝损伤更加敏感,而这一现象与肝脏NK细胞的异常活化相关,肝脏NK细胞发挥病理性免疫损伤作用主要是通过;NKG2D/NKG2DL(H60)相关信号通路及其胞内与其免疫活性相关的细胞因子(IFN-y、TNF-α等)的表达。本研究从肝脏天然免疫系统角度部分解释了HCV和肝脏免疫细胞间的相互关系,对HCV急慢性感染的防控及HCV导致的肝脏疾病的治疗具有一定的理论意义。而该动物模型的建立也为今后探索HCV介导的肝脏病理生理学机制及研发潜在的HCV疫苗及免疫治疗方法提供了一个有效的工具。
[Abstract]:Hepatitis C Virus ( HCV ) is a single strand RNA virus of the genus Flavivirus , which is one of the main pathogenic factors of chronic hepatitis . The clinical data show that the liver cells in the patients with hepatitis C are extremely vulnerable to injury , and the repair ability of the liver cells is weakened and the hyperplasia is abnormal .

HCV is a non - cytotoxic hepatitis B virus , which is caused by the interaction between the host immune system and the infected liver cells . In particular , the abnormal immune response of the immune system of the host liver to the infected liver cells leads to the imbalance of liver cell damage and repair . However , the mechanism of the interaction between the immune system of HCV and the host liver is not clear , and it is an important purpose of this study to explore the interaction between HCV and the immune system of the host liver .

The study is carried out in two parts :

1 . Exploration and application of liver - specific stable transfection technology

A suitable animal model is an important part of exploring the pathogenesis of HCV . At present , the animal model which can infect HCV is limited to the model of human liver cell mouse model and several kinds of HCV receptor combined transgenic mice model . However , the traditional HCV transgenic mouse model lays a good foundation for studying the interaction between HCV protein and host , but it can not be applied to the interaction between HCV and liver due to the existence of innate immune tolerance .

1.1 Establishment of dynamic transfection technique for subretinal vein plexus

In this study , we have established a new method for the transfection of HCV mouse model instead of HCV in vivo .

1.2 Codon - optimized phage integration enzyme - mediated foreign gene expression in mouse liver for a long term

The bacteriophage integration enzyme technology is a biological enzyme technology that can mediate the specific integration of foreign genes in target cells . The newly reported mouse codon optimized phage integration enzyme optimizes codon usage for mouse gene characteristics , and has higher integration efficiency for murine source cells .

In order to promote the long - term and stable expression of foreign genes in liver tissues by the transfection of hydrodynamic transfection , we introduced the technique of mouse codon - optimized phage integration in the experiment . Using NF - 魏B sensor plasmid as the reporter gene , we established a small animal model for long - term stable transfection of liver NF - 魏B by co - transfection with phage - integrated enzyme expression plasmid . The results showed that the phage - integrated enzyme optimized by codon codon can more effectively promote the integration of exogenous gene in liver cells of living mice and mediate long - term high - efficiency expression of foreign genes .

1.3 Establishment of a Mouse Model for Long - term Expression of HCV Genomic DNA Based on Water - powered transfection and Integration of Enzyme Technology

On the basis of previous research , we established an animal model of long - term stable expression of HCV whole genome based on the normal C57BL / 6 mice immunized with water power transfection technology and mouse codon . In addition , the expression of the reporter gene - Firefly luciferase and HCV was fused on the HCV genome expression vector , and the expression of HCV in the mouse liver can be monitored in real time through the living imaging technique , thus providing convenience for subsequent experiments .

2 . Mechanism of HCV Promoting Liver Immune Injury in Mice

NK cells , which account for 30 % of the total liver lymphocytes , are of great importance to the invasion of liver cells . However , it is not clear whether NK cells are involved in the immune process of liver injury in patients with hepatitis C .

2.1 Effects of HCV on immune cells in liver of mice

The effect of HCV expression on the immune cells of liver was first analyzed . The results showed that HCV decreased the number of NK cells in the liver , but did not influence the activity of HCV .
HCV has no significant effect on other types of liver lymphocytes ( NKT , T , DC , etc . ) in mice . This results are similar to clinical reports .

2.2 HCV Promoting Con A - induced Liver Injury in Mice and Its Immunological Mechanism

In view of the important role played by NK cells in the natural immune system of the liver , we believe that the reduction of NK cells in the liver may have some effect on the immune response of the organism . In order to further explore the relationship between the HCV and the immune system of the host liver , the mechanism of the immune pathogenesis of HCV is explored based on the model of the HCV mouse and the autoimmune liver injury model induced by Con A .
The results showed that the amount and function of NK cells in mice induced by Con A were the most obvious . The results showed that the sensitivity of NK cells to Con A - induced liver injury was lower than that in control group .

In conclusion , this study has established a visualized model of HCV liver - specific long - term expression in C57BL / 6 mice . The study shows that this model is more sensitive to Con A - induced liver injury , and this phenomenon is related to abnormal activation of NK cells in the liver .
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.63;R-332

【参考文献】