MHV-3诱导的暴发型肝衰竭小鼠模型中NK细胞趋化机制的研究

发布时间：2018-05-13 02:45

本文选题：NK细胞 + 暴发型肝衰竭　；参考：《华中科技大学》2012年硕士论文

【摘要】：【研究背景】在我国乙型肝炎病毒（HBV）感染所导致的慢加急性肝衰竭（ACLF）比例呈现上升趋势，该疾病病情危重，死亡率高达70%。目前对于乙肝病毒所致的慢加急性肝衰竭的发生发展机制比较明确的是，病毒和宿主的双重因素。病毒蛋白、基因型以及变异均可参与到疾病的发生之中，而宿主的部分基因多态性及免疫学因素也均参与该疾病的发生。目前对于HBV-ACLF的免疫学发病机制的研究较为丰富，国内外的研究结果显示抗原特异性细胞毒性T细胞（CTL）在肝脏损伤中发挥重要作用。我们前期研究首次在鼠三型肝炎病毒（MHV-3）诱导的暴发型肝衰竭小鼠模型中阐明了肝脏NK细胞的作用及机制，同时发现NK细胞具有向肝脏募集的趋势，杀伤病毒感染的靶细胞能力增强，在肝衰竭病程进展过程中发挥了重要作用。在上述研究模型基础上，我们亦利用基因芯片技术，筛选出具有差异表达的肝脏NK细胞趋化因子受体CCR1、CCR5、CXCR3和CXCR4（表1）。为此，本研究拟在上述基因芯片研究结果的基础上，继续探讨上述四种肝脏NK细胞趋化因子受体在MHV-3诱导的暴发型肝衰竭小鼠模型中趋化机制，为进一步阐述病毒诱导的肝衰竭肝损伤免疫学发病机制提供实验依据。【研究目的】 1.以MHV-3诱导的暴发型肝衰竭小鼠模型为研究对象，，基于前期基因芯片结果，研究肝脏NK细胞趋化因子受体CCR1、CCR5、CXCR3和CXCR4表达水平的变化。找到疾病进展过程中，肝脏NK细胞关键性的趋化因子受体，为后续疾病干预研究提供靶点。 2.以MHV-3诱导的暴发型肝衰竭小鼠模型为研究对象，基于NK细胞关键性的趋化因子受体的结果，找出与之相关的肝脏趋化因子表达水平的变化，阐述NK细胞趋化机制，同时为后续疾病干预研究提供靶点。【研究方法】 1.利用实时荧光定量PCR技术，观察MHV-3感染Babl/cJ小鼠后0h（基线）、24h、48h和72h的肝脏NK细胞趋化因子受体CCR1、CCR5、CXCR3和CXCR4mRNA水平变化。 2.根据以上找到的肝脏NK细胞上表达变化显著的趋化受体，通过实时荧光定量PCR技术，动态观察MHV-3感染0h（基线）、24h、48h和72h后Babl/cJ小鼠肝组织中相应趋化因子mRNA水平变化。【研究结果】 1.随MHV-3感染Balb/cJ小鼠时间延长，肝脏NK细胞表面趋化因子受体CCR1、CCR5和CXCR4的mRNA表达水平逐渐升高。CCR1和CXCR4的mRNA表达水平在24h达峰值，分别为基线（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即恢复到基线水平，分别约为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表达水平在感染后逐渐增高，24h（3.346±0.614，P0.05）即较基线水平有显著升高,48h达峰值为基线水平的5倍（5.059±0.350，P0.05），到72h恢复到基线水平。CXCR3的mRNA表达水平在感染后变化不显著，在24h为基线水平的1.15倍（1.150±0.179，P0.05），48h的表达水平为基线水平的0.7倍（0.686±0.055，P0.05）。 2.根据前面研究结果，我们选取趋化因子受体CCR5作为进一步的研究对象，检测其相关配体CCL3(MIP-1α)、CCL4(MIP-1β)和CCL5(RANTES)在肝脏组织中随感染时间延长的表达水平。结果显示CCL3、CCL4和CCL5的mRNA表达水平均呈现上升趋势，均在感染后72h达峰值，分别约是基线水平（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）。【研究结论】 1.通过对前期基因芯片筛选出的NK细胞相关趋化因子受体CCR1、CXCR3、CXCR4和CCR5表达水平的验证，CCR1、CCR5和CXCR4在感染后48h内肝脏NK的mRNA水平均显著上调，尤以CCR5在48h升高明显。 2.随感染时间延长，肝脏组织中NK细胞趋化因子受体CCR5的相关配体CCL3(MIP-1α)、CCL4(MIP-1β)和CCL5(RANTES)mRNA水平均显著升高。
[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 卤 0.713 , P0.05 ) and 6 - fold ( 6.008 卤 1.301 , P0.05 ) , respectively , and the levels of CCR1 and CXCR4 were respectively 0.8 - fold ( 0.804 卤 0.144 , P0.05 ) and 1.6 - fold ( 1.618 卤 0.207 , P0.05 ) . The level of mRNA expression was increased gradually after infection ( 3.346 卤 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伪 ) , 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 卤 24.143 , P0.05 ) , 249 - fold ( 248.9 卤 48.336 , P0.05 ) and 49 - fold ( 48.68 卤 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伪 , MIP - 1尾 and IL - 1 were significantly increased in liver tissues .

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R512.62

【参考文献】