本文選題：丙型病毒性肝炎 + 動物模型　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年博士論文

【摘要】：丙型肝炎病毒(Hepatitis C Virus, HCV)是黃病毒屬的單股正鏈RNA病毒,是慢性肝炎的主要致病因子之一。臨床資料顯示,丙肝患者的肝細胞極易發(fā)生損傷,且肝細胞的自身修復(fù)能力減弱、增生異常,最終引發(fā)肝纖維化、肝硬化及肝癌的發(fā)生,迄今仍缺乏有效的防治手段。加強丙型病毒性肝炎的相關(guān)基礎(chǔ)研究,是當前丙型病毒性肝炎防治研究中的重要命題。 HCV是一種非細胞毒性的嗜肝病毒,丙肝患者肝細胞極易發(fā)生損傷的現(xiàn)象主要是由宿主免疫系統(tǒng)與受病毒感染的肝細胞間的相互作用引起。特別是宿主肝臟免疫系統(tǒng)對被感染肝實質(zhì)細胞的異常免疫反應(yīng)導(dǎo)致了肝細胞損傷與修復(fù)的平衡失調(diào)。但HCV和宿主肝臟免疫系統(tǒng)間相互作用的機制尚不明確,探索HCV和宿主肝臟免疫系統(tǒng)之間的相互作用是本研究的重要目的。 本研究分以下兩個部分展開： 1.肝臟特異性穩(wěn)定轉(zhuǎn)染技術(shù)的探索與應(yīng)用 合適的動物模型是探索HCV免疫致病機理的重要環(huán)節(jié)。目前,可感染HCV的動物模型僅限于黑猩猩、嵌合人類肝細胞小鼠模型及多種HCV受體聯(lián)合轉(zhuǎn)基因小鼠模型。但由于受數(shù)量有限、價格昂貴等因素的影響,限制了這幾類動物模型在基礎(chǔ)研究中的廣泛應(yīng)用。傳統(tǒng)的HCV轉(zhuǎn)基因小鼠模型為研究HCV蛋白與宿主之間的相互作用奠定了良好的基礎(chǔ),但由于存在先天免疫耐受,使其不能應(yīng)用于HCV和肝臟免疫系統(tǒng)間的相互作用研究。基于此,采用合適的方法建立免疫功能正常并適于實驗室廣泛應(yīng)用的HCV動物模型是目前抗HCV相關(guān)研究的重要環(huán)節(jié)。 1.1眼底靜脈叢水動力轉(zhuǎn)染技術(shù)的建立 基于經(jīng)典的水動力轉(zhuǎn)染技術(shù)和眼底靜脈叢注射技術(shù)建立的眼底靜脈叢水動力轉(zhuǎn)染技術(shù)具有良好的肝臟靶向性,在目前缺乏合適的HCV小動物模型的前提下,通過該方法建立HCV小鼠模型替代HCV體內(nèi)感染模型可能是一種快速、高效、穩(wěn)定、簡便的構(gòu)建HCV肝臟特異性轉(zhuǎn)基因動物模型的新方法。傳統(tǒng)的水動力轉(zhuǎn)染技術(shù)有其自身難以克服的缺點,因此,本研究結(jié)合眼底靜脈叢輸注建立了眼底靜脈叢水動力轉(zhuǎn)染技術(shù)。該技術(shù)是將大量含有目的基因表達質(zhì)粒的鹽水溶液從眼底靜脈快速注入實驗動物體內(nèi),實現(xiàn)外源基因在肝臟特異、高效表達。通過條件摸索,確立了應(yīng)用該技術(shù)的最適轉(zhuǎn)染條件,并分析了其對實驗動物器官機能的影響。在轉(zhuǎn)染效率方面,眼底靜脈叢水動力轉(zhuǎn)染技術(shù)與經(jīng)典的尾靜脈水動力轉(zhuǎn)染技術(shù)相當。該技術(shù)操作更簡單、可重復(fù)性強。最為關(guān)鍵的是,眼底靜脈叢水動力轉(zhuǎn)染技術(shù)克服了尾靜脈水動力轉(zhuǎn)染僅能用于尾靜脈適合注射的實驗動物的應(yīng)用限制。該技術(shù)除可用于常規(guī)實驗動物外,還可用于尾靜脈無法注射或無尾靜脈的實驗動物,有效拓展了水動力轉(zhuǎn)染技術(shù)的應(yīng)用范圍。 1.2密碼子優(yōu)化的噬菌體整合酶介導(dǎo)外源基因在小鼠肝臟長期穩(wěn)定表達 噬菌體整合酶技術(shù)是一種可以介導(dǎo)外源基因在靶細胞發(fā)生特異性整合的生物酶技術(shù)。新近報道的鼠密碼子優(yōu)化的噬菌體整合酶針對鼠類基因特點進行密碼子優(yōu)化,對于鼠源細胞具有更高的整合效率。 為促進水動力轉(zhuǎn)染技術(shù)導(dǎo)入肝組織中的外源基因在小鼠肝細胞中長期穩(wěn)定表達,我們在實驗中引入鼠密碼子優(yōu)化的噬菌體整合酶技術(shù)。并以NF-κB sensor質(zhì)粒作為報告基因,通過與噬菌體整合酶表達質(zhì)粒共轉(zhuǎn)染實現(xiàn)對活體小鼠肝臟長期穩(wěn)定轉(zhuǎn)染的技術(shù)優(yōu)化,建立了可長期監(jiān)測肝臟NF-κB活性的小動物模型。研究結(jié)果顯示,與傳統(tǒng)的噬菌體整合酶系統(tǒng)相比,鼠密碼子優(yōu)化的噬菌體整合酶可以更加有效的促使外源基因在活體小鼠肝細胞中發(fā)生整合,并介導(dǎo)外源基因長期高效表達。 1.3基于水動力轉(zhuǎn)染和整合酶技術(shù)建立HCV基因組肝臟長期表達小鼠模型 在前期研究工作基礎(chǔ)上,我們聯(lián)合水動力轉(zhuǎn)染技術(shù)和鼠密碼子優(yōu)化的噬菌體整合酶技術(shù),基于免疫功能正常的C57BL/6小鼠,建立了HCV全基因組肝臟長期穩(wěn)定表達的動物模型。此外,在HCV全基因組表達載體上融合了報告基因—螢火蟲熒光素酶,HCV在小鼠肝臟的表達情況可以通過活體成像技術(shù)進行實時監(jiān)測,為后續(xù)實驗提供了方便。 2.HCV促進小鼠肝臟免疫損傷的機制研究 強勢的肝臟免疫系統(tǒng)是機體抵御外來侵襲的重要防御機制。占肝臟淋巴細胞總數(shù)30%左右的NK細胞,對抵抗病毒、寄生菌感染和惡性轉(zhuǎn)化細胞侵潤等侵害具有十分重要的意義。但NK細胞是否參與丙肝患者肝細胞損傷的免疫過程尚不明確。因此本研究尋找以NK細胞為代表的肝臟免疫細胞在丙肝患者肝細胞損傷中的作用機制,可能是防治HCV所導(dǎo)致的肝臟疾病的重要方法。 2.1HCV對小鼠肝臟免疫細胞的影響 以本研究建立的免疫功能正常的HCV全基因組肝臟長期表達小鼠模型為基礎(chǔ),首先分析HCV表達對肝臟免疫細胞的影響,結(jié)果顯示：HCV降低了小鼠肝臟NK細胞的數(shù)量,但不影響其活性；HCV對小鼠其他類型的肝臟淋巴細胞(NKT、T、DC等)未造成顯著影響。這一結(jié)果與臨床報道相似。 2.2HCV促進Con A誘導(dǎo)的小鼠肝臟損傷及其免疫學(xué)機制 鑒于NK細胞在肝臟天然免疫系統(tǒng)中發(fā)揮的重要作用,我們認為,肝臟NK細胞的減少可能會對機體的免疫反應(yīng)產(chǎn)生一定的影響。為進一步探索HCV與宿主肝臟免疫系統(tǒng)的關(guān)系,基于該HCV小鼠模型,并結(jié)合Con A誘導(dǎo)的自身免疫性肝損傷模型,對HCV免疫致病機理進行探索。結(jié)果顯示：中等劑量Con A刺激后,HCV組小鼠的肝臟免疫損傷現(xiàn)象增強；而高劑量Con A刺激后,HCV小鼠發(fā)生嚴重的自身免疫性肝炎并促使小鼠死亡率增高。進一步研究結(jié)果顯示：ConA靜脈注射誘發(fā)小鼠自身免疫性肝炎后,小鼠肝臟免疫細胞活化,其中肝臟天然免疫細胞---NK細胞的數(shù)量和功能在HCV組和對照組小鼠間的差異最為明顯。選擇性清除小鼠體內(nèi)的NK細胞,則HCV小鼠對Con A誘導(dǎo)的肝損傷的敏感性降低,肝損傷減輕。說明HCV小鼠對Con A誘導(dǎo)的肝損傷的敏感性增強與小鼠肝臟NK細胞的過度活化相關(guān)。針對NK細胞功能的研究結(jié)果顯示,肝臟NK細胞免疫功能過度激活依賴于NK細胞表面激活性受體NKG2D及其胞內(nèi)與其免疫活性密切相關(guān)的細胞因子(IFN-γ、TNF-α等)的表達。同時,NKG2D在肝細胞表面的配體NKG2DL(H60)的表達水平也顯著上調(diào)。 綜上所述,本研究結(jié)合眼底靜脈叢水動力轉(zhuǎn)染技術(shù)、密碼子優(yōu)化的噬菌體整合酶和活體熒光成像技術(shù),基于免疫功能正常的C57BL/6小鼠,建立了可視化的HCV肝臟特異性長期表達小鼠模型。研究顯示,該模型對Con A誘導(dǎo)的肝損傷更加敏感,而這一現(xiàn)象與肝臟NK細胞的異�；罨嚓P(guān),肝臟NK細胞發(fā)揮病理性免疫損傷作用主要是通過;NKG2D/NKG2DL(H60)相關(guān)信號通路及其胞內(nèi)與其免疫活性相關(guān)的細胞因子(IFN-y、TNF-α等)的表達。本研究從肝臟天然免疫系統(tǒng)角度部分解釋了HCV和肝臟免疫細胞間的相互關(guān)系,對HCV急慢性感染的防控及HCV導(dǎo)致的肝臟疾病的治療具有一定的理論意義。而該動物模型的建立也為今后探索HCV介導(dǎo)的肝臟病理生理學(xué)機制及研發(fā)潛在的HCV疫苗及免疫治療方法提供了一個有效的工具。
[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 .
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R512.63;R-332

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本文編號：2110696