日本血吸虫感染小鼠肝脏内枯否细胞的动态分析及其功能初步研究

发布时间：2018-05-29 12:31

本文选题：S.japonicum + 枯否细胞　；参考：《第二军医大学》2017年硕士论文

【摘要】：血吸虫病(Schistosomiasis)是一种被严重忽视的热带疾病,威胁全球约2亿人的健康。感染人体的血吸虫有5种,在我国,只有日本血吸虫(Schistosoma japonicum,S.japonicum)流行,主要分布于长江中游及下游流域的湖泊和沼泽地区。日本血吸虫感染终宿主后,其在宿主体内经历了不同生活史发育阶段,并可引起宿主的病理反应及造成损害。特别是当日本血吸虫虫卵大量沉积在肝脏时,可引起肝脏肉芽肿,进而引起肝脏纤维化,严重危害患者的健康。在日本血吸虫引起宿主肝脏炎症和纤维化的过程中,肝脏内的巨噬细胞发挥了重要作用。在血吸虫病急性期,巨噬细胞以经典活化为主,在肝脏内引起严重的炎症反应。在血吸虫病进入慢性期时,巨噬细胞以旁路活化为主,能够对宿主产生抗炎的保护作用,并促进纤维化的形成。肝脏中有一类起源于胚胎卵黄囊,只能依靠自我增殖进行更新补充的巨噬细胞,被称为枯否细胞(Kupffer cells,KCs)。KCs在肝窦不断逡巡,能够吞噬进入肝脏中的外界抗原物质,是保护肝脏的第一道防线。目前尚不清楚KCs在日本血吸虫致病过程中的作用。近年来,微小RNA(micro RNA,miRNA)是一个饱受关注的研究领域。miRNA是内源性19~25个碱基的小片段非编码RNA。miRNA可结合到靶基因3’端非翻译区(3’UTR),抑制靶基因m RNA的翻译或者导致靶基因m RNA降解,从而参与调控多种疾病的病理过程,如肿瘤发生、病毒感染、纤维化形成等。本研究首先检测了小鼠感染日本血吸虫前后其肝脏KCs的动态变化;在此基础上,我们筛选日本血吸虫感染后小鼠肝脏KCs内差异表达的miRNA及m RNA,并采用KEGG软件,分析这些差异表达基因的信号通路;最后对其中的一个差异表达的miRNA及其靶基因进行实验研究。首先,我们利用密度梯度离心和流式分选相结合的方法,获得正常组和感染血吸虫组小鼠的KCs。在流式分选的过程中,我们发现日本血吸虫感染后不同时间,宿主肝脏内的非实质细胞(non-parenchyma cells,NPCs)存在动态变化。我们判定F4/80~(hi) CD11b~(lo) NPCs细胞为肝脏KCs;F4/80lo CD11bhi表型细胞为血液中募集进入肝脏的NPCs。在小鼠感染日本血吸虫的过程中,我们研究了这两群细胞的动态变化:对照组的正常小鼠中,肝脏内仅有F4/80~(hi) CD11b~(lo)表型的KCs,且数量稳定,在肝脏NPCs中的比例维持在20%左右;而感染日本血吸虫的小鼠,其F4/80~(hi) CD11b~(lo) KCs的比例开始为20%左右,而随着血吸虫病的进程,该KCs逐渐减少至少于1%。与此同时,从血液募集的F4/80lo CD11bhi NPCs细胞群,在感染4周后出现,其数量急剧增加并在感染后期保持稳定。肝脏内NPCs随着日本血吸虫的感染时间而动态变化,提示我们在日本血吸虫的不同致病阶段中,可能有不同的非实质细胞参与其中,发挥相应的功能。为了进一步探讨F4/80~(hi) CD11b~(lo) KCs在日本血吸虫感染过程逐渐减少的内在因素,我们采用miRNA芯片和深度测序的方法,分析日本血吸虫感染前后宿主KCs miRNA和m RNA表达谱,并鉴定出差异表达的miRNA和m RNA。实验取日本血吸虫感染后第6周的小鼠以及正常对照组小鼠,分离两组小鼠肝脏的F4/80~(hi) CD11b~(lo) KCs样本,提取miRNA和总RNA,分别进行miRNA芯片检测和转录本测序。miRNA芯片检测结果经分析显示:共获得63个差异表达3倍以上的miRNA,其中上调表达miRNA 51个,下调表达miRNA 12个。随机挑选其中20个差异表达的miRNA进行实验验证,经实时荧光定量PCR(quantitative real time PCR,q RT-PCR)验证,其中17个和芯片检测结果趋势一致。通过转录本测序和分析,我们从中筛选到差异表达2倍以上的基因共1083个,其中上调表达494个,下调表达589个。采用KEGG软件分析,这些差异表达基因分属于T细胞受体信号通路、趋化因子信号通路等10个信号通路。选取趋化因子相关通路进行q RT-PCR验证,其结果和转录本测序结果趋势一致。我们进一步在感染后4至9周不同时间点的感染组和对照组样本中检测CCL3、CCL4、CCL7等趋化因子,结果显示,这些趋化因子在感染后不同时间点均上调表达,提示我们日本血吸虫感染后,肝脏内NPCs的动态变化可能与趋化因子的增加有关。在临床研究中发现,肝癌病人KCs中,CX3CR1的表达下调。同时,肝脏内KCs中抗凋亡基因Bcl2表达下调,细胞有凋亡的趋势。在四氯化碳(CCL4)诱导小鼠肝纤维化模型的研究中,当CX3CR1敲除后,小鼠肝脏的纤维化更加严重。趋化因子受体CX3CR1能够介导胞外促生存信号传递到胞内,也能够影响肝脏的纤维化,但是二者之间是否存在关联、其中的具体机制目前尚不明确。从转录本测序结果中我们发现,日本血吸虫感染后趋化因子受体CX3CR1的表达与正常小鼠相比有显著下降。与此同时,我们发现感染后小鼠肝脏KCs中mi R-297a-5p显著升高。通过在线靶基因预测,在CX3CR1基因的3’端非翻译区(untranslated region)存在mi R-297a-5p的作用靶点。因此,我们对CX3CR1在日本血吸虫感染后F4/80~(hi) CD11b~(lo) KCs减少中的作用以及与mi R-297a-5p的靶向关系开展了进一步研究。我们采用q RT-PCR方法,检测日本血吸虫感染后不同时间点感染组和对照组F4/80~(hi) CD11b~(lo) KCs RNA样本,结果显示感染后CX3CR1的表达呈显著下调,mi R-297a-5p表达逐渐增加。此外,感染组和对照组F4/80~(hi) CD11b~(lo) KCs表面CX3CR1流式分析结果显示,感染组KCs表面CX3CR1蛋白表达下调,而对照组CX3CR1蛋白表达没有显著变化。为验证CX3CR1与mi R-297a-5p之间的靶向关系,我们利用双荧光报告系统进行了检测。首先,我们依据在线预测的靶向结合位点,合成了野生型CX3CR1 3’UTR结合位点和突变型结合位点,并插入到带有荧光基因的报告载体上。将mi R-297a-5p模拟物(mimics)、野生型和突变型报告载体分别转染到HEK-293T细胞中,培养48小时。经检测,mi R-297a-5p高表达后,野生型CX3CR1 3’UTR载体报告基因的荧光表达会显著下调(p0.05),但是突变型CX3CR1 3’UTR载体报告基因的表达没有显著(p0.05)的变化。为进一步研究KCs内差异表达的mi R-297a-5p及其靶基因CX3CR1在日本血吸虫致病过程中的作用,我们利用巨噬细胞系,在体外对mi R-297a-5p及CX3CR1进行了初步的功能研究。我们利用电转染的方法,将不同浓度的mi R-297a-5p mimics分别转染到KCs细胞系和RAW264.7细胞系中。q RT-PCR检测,mi R-297a-5p表达量显著提高,并能够有效抑制CX3CR1 m RNA的表达。进一步利用流式检测的方法,对转染细胞表面CX3CR1蛋白表达以及凋亡情况进行检测。检测结果显示,细胞表面CX3CR1蛋白表达无变化,而且也并未呈现凋亡的现象。在本课题的研究中,我们发现日本血吸虫感染后,肝脏内NPCs动态变化。其动态变化与日本血吸虫致病过程有紧密关联。我们筛选出了日本血吸虫感染后KCs中差异表达的miRNA和m RNA。对mi R-297a-5p和CX3CR1的靶向关系及功能进行了体外初步研究。本课题为进一步研究KCs在日本血吸虫致病过程中的作用奠定了基础。
[Abstract]:Schistosomiasis (Schistosomiasis) is a seriously neglected tropical disease that threatens the health of about 200 million people around the world. There are 5 kinds of Schistosoma infected human Schistosoma. In our country, only Schistosoma japonicum (S.japonicum) is prevalent in lakes and swamps in the middle and lower reaches of the Yangtze River. After host, it has undergone different life history stages in the host, and can cause pathological reaction and damage to the host. Especially, when the eggs of Schistosoma japonicum are deposited in the liver, it can cause liver granuloma, and then cause liver fibrosis and seriously harm the health of the patients. In the process of fibrosis, macrophages play an important role in the liver. In the acute phase of schistosomiasis, macrophages are mainly activated by classical activation, causing severe inflammatory reactions in the liver. When schistosomiasis enters the chronic phase, macrophages are mainly by-pass activation, which can produce anti-inflammatory protective effects to the host and promote fibrosis. Formation. In the liver, there is a type of macrophage that originates from the egg yolk sac, which can only be supplemented by self proliferation. It is called Kupffer cells (KCs).KCs in the hepatic sinus. It is the first defense line to protect the liver. It is not clear at present that KCs is caused by Schistosoma japonicum. The role of the disease process. In recent years, the small RNA (micro RNA, miRNA) is a hot research field..miRNA is a small fragment of endogenous 19~25 base, a small fragment of non coded RNA.miRNA can bind to the target gene 3 'end non translation region (3' UTR), inhibit the translation of the target gene m RNA or lead to the target gene m RNA degradation, thus participating in the regulation of a variety of diseases. In this study, we first detected the dynamic changes of KCs in the liver of mice infected with Schistosoma japonicum. On this basis, we screened the differential expression of miRNA and m RNA in the liver KCs of mice infected with Schistosoma japonicum, and the KEGG software was used to analyze these differentially expressed genes. In the end, we used the method of combining the density gradient centrifugation and the flow separation to obtain the KCs. in the normal group and the infected schistosomiasis group in the flow sorting process. We found that the host of Schistosoma japonicum was infected at different time and the host at different time. There are dynamic changes in the non-parenchyma cells (NPCs) in the liver. We determine that F4/80~ (HI) CD11b~ (LO) NPCs cells are liver KCs; F4/80lo CD11bhi phenotypic cells recruit the liver into the liver in the course of mice infected with Schistosoma japonicum. We studied the dynamic changes of these two groups of cells: the control group was positive. In normal mice, only the KCs of F4/80~ (HI) CD11b~ (LO) phenotype in the liver is stable and the proportion in the liver NPCs is maintained at about 20%, while the proportion of F4/80~ (HI) CD11b~ (LO) KCs in mice infected with Schistosoma japonicum begins to be about 20%, and with the process of blood sucking, the KCs gradually decreases to less than that of the blood. The number of F4/80lo CD11bhi NPCs cells in the set, which appeared after 4 weeks of infection, increased rapidly and remained stable at the later stage of infection. The NPCs in the liver changed dynamically with the infection time of Schistosoma japonicum, suggesting that we may have different non parenchymal cells involved in the different pathogenic stages of Schistosoma japonicum, and the corresponding function is played. In order to further explore the intrinsic factors of the gradual decrease of F4/80~ (HI) CD11b~ (LO) KCs in the infection process of Schistosoma japonicum, we used miRNA chips and deep sequencing methods to analyze the expression profiles of KCs miRNA and m RNA of the host before and after infection of Schistosoma japonicum, and identified the miRNA and M experiments for sixth weeks after the infection of Schistosoma japonicum. In mice and normal control mice, the F4/80~ (HI) CD11b~ (LO) KCs samples of the liver of two groups were isolated and miRNA and total RNA were extracted. The results of.MiRNA chip detection and transcriptional sequencing of the transcriptional transcriptional sequences were analyzed. The results showed that 63 differential expressions of 3 times of miRNA were obtained, among which 51 were up to up expression, and the expression miRNA 12 was down. A random selection of 20 differentially expressed miRNA was tested and verified by real time fluorescence quantitative PCR (quantitative real time PCR, Q RT-PCR), and 17 of them were consistent with the trend of chip detection results. Through transcriptional sequencing and analysis, we screened 1083 genes with more than 2 times of differential expression, of which 494 were up-regulated. Down regulated expression of 589. Using KEGG software, these differentially expressed genes were divided into 10 signal pathways, such as T cell receptor signaling pathway, chemokine signaling pathway and other signaling pathways. The chemokine related pathway was selected for Q RT-PCR validation, and the results were consistent with the trend of the transcriptional sequencing results. We further felt the sense of time points at 4 to 9 weeks after infection. CCL3, CCL4, CCL7 and other chemokines were detected in the samples of the dyed and control groups. The results showed that these chemokines were up-regulated at different time points after infection, suggesting that the dynamic changes of NPCs in the liver may be related to the increase of chemokine in the liver after infection of Schistosoma japonicum. In clinical study, the expression of CX3CR1 in the KCs of the patients with liver cancer was found. Down regulation. At the same time, the expression of anti apoptotic gene Bcl2 in the liver of KCs is down, and the cells have the trend of apoptosis. In the study of the liver fibrosis model induced by carbon tetrachloride (CCL4), when the CX3CR1 knocks out, the liver fibrosis is more serious in mice. Chemokine receptor CX3CR1 can mediate the extracellular promoting survival signal to the intracellular, and can affect the liver. It is not clear whether there is an association between the two, but the specific mechanism is not clear at the moment. From the transcriptional sequence we found that the expression of chemokine receptor CX3CR1 after Schistosoma japonicum infection was significantly lower than that in normal mice. At the same time, we found that the MI R-297a-5p in the KCs of infected mice liver is significant. The target of MI R-297a-5p exists in the 3 'end non translation region (untranslated region) of the CX3CR1 gene. Therefore, we further studied the role of CX3CR1 in the F4/80~ (HI) CD11b~ (LO) KCs decrease after CX3CR1 infection and the relationship with the targeting of MI. The R method was used to detect F4/80~ (HI) CD11b~ (LO) KCs RNA samples in the infection group and the control group after the infection of Schistosoma japonicum. The results showed that the expression of CX3CR1 was significantly down after infection and the expression of MI R-297a-5p increased gradually. The expression of CX3CR1 protein was down, but the expression of CX3CR1 protein in the control group was not significantly changed. In order to verify the targeting relationship between CX3CR1 and MI R-297a-5p, we used the dual fluorescence report system to detect it. First, we synthesized the wild type CX3CR1 3 'UTR binding site and the mutant binding site based on the targeted binding site of the online CX3CR1. Mi R-297a-5p mimics (mimics), wild type and mutant report vectors were transfected into HEK-293T cells for 48 hours respectively. After detection, the high expression of MI R-297a-5p, the fluorescent expression of the wild type CX3CR1 3 'UTR carrier reporter gene could be significantly downregulated (P0.05), but mutated CX3CR1 There is no significant (P0.05) change in the expression of the 3 'UTR carrier reporter gene. To further study the role of MI R-297a-5p and its target gene CX3CR1 in the pathogenesis of Schistosoma japonicum, we use macrophage system to carry out preliminary functional studies on MI R-297a-5p and CX3CR1 in vitro. We use electrotransfection prescription in vitro. The MI R-297a-5p mimics of different concentrations was transfected to.Q RT-PCR in KCs cell line and RAW264.7 cell line respectively. The R-297a-5p expression of MI was significantly increased and the expression of CX3CR1 m RNA was inhibited effectively. The expression of protein and apoptosis of the transfected cells were detected by flow detection. The results showed that there was no change in the expression of CX3CR1 protein on the surface of the cell, and there was no apoptosis. In this study, we found the dynamic changes of NPCs in the liver after the infection of Schistosoma japonicum. The dynamic changes were closely related to the pathogenesis of Schistosoma japonicum. We screened the differential expression in KCs after Schistosoma japonicum infection. The target relationship and function of MI R-297a-5p and CX3CR1 were preliminarily studied by miRNA and m RNA. in vitro. This topic laid the foundation for further study of the role of KCs in the pathogenesis of Schistosoma japonicum.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R532.21

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