本文選題：NK細胞 + 暴發(fā)型肝衰竭��； 參考：《華中科技大學(xué)》2012年碩士論文

【摘要】：【研究背景】 在我國乙型肝炎病毒（HBV）感染所導(dǎo)致的慢加急性肝衰竭（ACLF）比例呈現(xiàn)上升趨勢，該疾病病情危重，死亡率高達70%。目前對于乙肝病毒所致的慢加急性肝衰竭的發(fā)生發(fā)展機制比較明確的是，病毒和宿主的雙重因素。病毒蛋白、基因型以及變異均可參與到疾病的發(fā)生之中，而宿主的部分基因多態(tài)性及免疫學(xué)因素也均參與該疾病的發(fā)生。 目前對于HBV-ACLF的免疫學(xué)發(fā)病機制的研究較為豐富，國內(nèi)外的研究結(jié)果顯示抗原特異性細胞毒性T細胞（CTL）在肝臟損傷中發(fā)揮重要作用。我們前期研究首次在鼠三型肝炎病毒（MHV-3）誘導(dǎo)的暴發(fā)型肝衰竭小鼠模型中闡明了肝臟NK細胞的作用及機制，同時發(fā)現(xiàn)NK細胞具有向肝臟募集的趨勢，殺傷病毒感染的靶細胞能力增強，在肝衰竭病程進展過程中發(fā)揮了重要作用。 在上述研究模型基礎(chǔ)上，我們亦利用基因芯片技術(shù)，篩選出具有差異表達的肝臟NK細胞趨化因子受體CCR1、CCR5、CXCR3和CXCR4（表1）。為此，本研究擬在上述基因芯片研究結(jié)果的基礎(chǔ)上，繼續(xù)探討上述四種肝臟NK細胞趨化因子受體在MHV-3誘導(dǎo)的暴發(fā)型肝衰竭小鼠模型中趨化機制，為進一步闡述病毒誘導(dǎo)的肝衰竭肝損傷免疫學(xué)發(fā)病機制提供實驗依據(jù)。 【研究目的】 1.以MHV-3誘導(dǎo)的暴發(fā)型肝衰竭小鼠模型為研究對象，，基于前期基因芯片結(jié)果，研究肝臟NK細胞趨化因子受體CCR1、CCR5、CXCR3和CXCR4表達水平的變化。找到疾病進展過程中，肝臟NK細胞關(guān)鍵性的趨化因子受體，為后續(xù)疾病干預(yù)研究提供靶點。 2.以MHV-3誘導(dǎo)的暴發(fā)型肝衰竭小鼠模型為研究對象，基于NK細胞關(guān)鍵性的趨化因子受體的結(jié)果，找出與之相關(guān)的肝臟趨化因子表達水平的變化，闡述NK細胞趨化機制，同時為后續(xù)疾病干預(yù)研究提供靶點。 【研究方法】 1.利用實時熒光定量PCR技術(shù)，觀察MHV-3感染Babl/cJ小鼠后0h（基線）、24h、48h和72h的肝臟NK細胞趨化因子受體CCR1、CCR5、CXCR3和CXCR4mRNA水平變化。 2.根據(jù)以上找到的肝臟NK細胞上表達變化顯著的趨化受體，通過實時熒光定量PCR技術(shù)，動態(tài)觀察MHV-3感染0h（基線）、24h、48h和72h后Babl/cJ小鼠肝組織中相應(yīng)趨化因子mRNA水平變化。 【研究結(jié)果】 1.隨MHV-3感染Balb/cJ小鼠時間延長，肝臟NK細胞表面趨化因子受體CCR1、CCR5和CXCR4的mRNA表達水平逐漸升高。CCR1和CXCR4的mRNA表達水平在24h達峰值，分別為基線（0h）水平的4倍（3.946±0.713，P0.05）和6倍（6.008±1.301，P0.05），感染后48h即恢復(fù)到基線水平，分別約為0h的0.8倍（0.804±0.144，P0.05）和1.6倍（1.618±0.207，P0.05）。而CCR5的mRNA表達水平在感染后逐漸增高，24h（3.346±0.614，P0.05）即較基線水平有顯著升高,48h達峰值為基線水平的5倍（5.059±0.350，P0.05），到72h恢復(fù)到基線水平。CXCR3的mRNA表達水平在感染后變化不顯著，在24h為基線水平的1.15倍（1.150±0.179，P0.05），48h的表達水平為基線水平的0.7倍（0.686±0.055，P0.05）。 2.根據(jù)前面研究結(jié)果，我們選取趨化因子受體CCR5作為進一步的研究對象，檢測其相關(guān)配體CCL3(MIP-1α)、CCL4(MIP-1β)和CCL5(RANTES)在肝臟組織中隨感染時間延長的表達水平。結(jié)果顯示CCL3、CCL4和CCL5的mRNA表達水平均呈現(xiàn)上升趨勢，均在感染后72h達峰值，分別約是基線水平（0h）的116倍（116.5±24.143，P0.05）、249倍（248.9±48.336，P0.05）和49倍（48.68±10.370，P0.05）。 【研究結(jié)論】 1.通過對前期基因芯片篩選出的NK細胞相關(guān)趨化因子受體CCR1、CXCR3、CXCR4和CCR5表達水平的驗證，CCR1、CCR5和CXCR4在感染后48h內(nèi)肝臟NK的mRNA水平均顯著上調(diào)，尤以CCR5在48h升高明顯。 2.隨感染時間延長，肝臟組織中NK細胞趨化因子受體CCR5的相關(guān)配體CCL3(MIP-1α)、CCL4(MIP-1β)和CCL5(RANTES)mRNA水平均顯著升高。
[Abstract]:BACKGROUND OF THE STUDY

The rate of acute liver failure ( ACLF ) caused by hepatitis B virus ( HBV ) infection in China presents a rising trend . The disease is critical and the mortality rate is as high as 70 % . At present , the pathogenesis of chronic hepatitis B virus caused by hepatitis B virus has a clear development mechanism . The virus protein , genotype and variation can be involved in the occurrence of the disease , and the partial gene polymorphism and immunological factors of the host are also involved in the occurrence of the disease .

At present , the study of the mechanism of immunological pathogenesis of HBV - ACLF is rich , and the research results show that the antigen - specific cytotoxic T - cell ( CTL ) plays an important role in the liver injury .

On the basis of the above research model , we also used gene chip technology to screen the chemokine receptor CCR1 , CCR3 and CXCR4 ( Table 1 ) of the liver NK cells with different expression . To this end , we intend to explore the mechanism of chemokine receptor chemokine receptor in the mouse model induced by MHV - 3 on the basis of the research results of the above - mentioned gene chip , and provide experimental basis for further elaboration of the mechanism of immunological pathogenesis of liver failure induced by virus .

Purpose of research

1 . MHV - 3 - induced liver failure mice model was used as the research object . Based on the results of early gene chip , the changes of the expression level of CCR1 , CCR3 and CXCR4 in the NK cells of the liver were studied . In the course of disease progression , the key chemokine receptors in the NK cells of the liver were found , which provided the target for the study of subsequent disease intervention .

2 . The mouse model induced by MHV - 3 was used as the research object . Based on the results of the key chemokine receptor in NK cells , the changes of the expression level of the liver chemokine related to it were found , the NK cell evolution mechanism was expounded , and the target was provided for the subsequent disease intervention study .

Methodology of research

1 . Using real - time fluorescence quantitative PCR technique , the levels of CCR1 , CCR1 , CCR1 , CCR3 and CCR4 mRNA in liver NK cells at 0 h ( baseline ) , 24 h , 48 h and 72 h after infection with MHV - 3 infected Babl / cJ mice were observed .

2 . The mRNA level of corresponding chemokine mRNA was observed in the liver tissues of Babl / cJ mice after 0 h ( baseline ) , 24 h , 48 h and 72 h after MHV - 3 infection by real - time fluorescence quantitative PCR .

Outcome of the study

1 . With MHV - 3 infected Balb / cJ mice , the mRNA expression levels of CCR1 and CXCR4 in liver NK cells increased gradually . The mRNA expression levels of CCR1 and CXCR4 were 4 - fold ( 3.946 鹵 0.713 , P0.05 ) and 6 - fold ( 6.008 鹵 1.301 , P0.05 ) , respectively , and the levels of CCR1 and CXCR4 were respectively 0.8 - fold ( 0.804 鹵 0.144 , P0.05 ) and 1.6 - fold ( 1.618 鹵 0.207 , P0.05 ) . The level of mRNA expression was increased gradually after infection ( 3.346 鹵 0.614 , P0.05 ) .

2 . Based on the results of the previous study , we selected chemokine receptor CCR3 as a further research object to detect the expression level of its associated ligand CClA ( MIP - 1偽 ) , CCL _ 4 ( MIP - 1尾 ) and CCL _ 5 in liver tissue . The results showed that the levels of mRNA expression in liver tissues were 116 - fold ( 116.5 鹵 24.143 , P0.05 ) , 249 - fold ( 248.9 鹵 48.336 , P0.05 ) and 49 - fold ( 48.68 鹵 10.370 , P0.05 ) .

Conclusions of the research

1 . The levels of CCR1 , CCR1 , CXCR4 and CCR1 in NK cells screened by early gene chip showed that CCR1 , CCR1 , CXCR4 and CXCR4 were significantly higher in liver NK mRNA levels in 48 hours after infection , especially in 48 hours after infection .

2 . As the time of infection was prolonged , the mRNA levels of MIP - 1偽 , MIP - 1尾 and IL - 1 were significantly increased in liver tissues .

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R-332;R512.62

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