本文選題：S.japonicum + 枯否細(xì)胞��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：血吸蟲病(Schistosomiasis)是一種被嚴(yán)重忽視的熱帶疾病,威脅全球約2億人的健康。感染人體的血吸蟲有5種,在我國,只有日本血吸蟲(Schistosoma japonicum,S.japonicum)流行,主要分布于長江中游及下游流域的湖泊和沼澤地區(qū)。日本血吸蟲感染終宿主后,其在宿主體內(nèi)經(jīng)歷了不同生活史發(fā)育階段,并可引起宿主的病理反應(yīng)及造成損害。特別是當(dāng)日本血吸蟲蟲卵大量沉積在肝臟時(shí),可引起肝臟肉芽腫,進(jìn)而引起肝臟纖維化,嚴(yán)重危害患者的健康。在日本血吸蟲引起宿主肝臟炎癥和纖維化的過程中,肝臟內(nèi)的巨噬細(xì)胞發(fā)揮了重要作用。在血吸蟲病急性期,巨噬細(xì)胞以經(jīng)典活化為主,在肝臟內(nèi)引起嚴(yán)重的炎癥反應(yīng)。在血吸蟲病進(jìn)入慢性期時(shí),巨噬細(xì)胞以旁路活化為主,能夠?qū)λ拗鳟a(chǎn)生抗炎的保護(hù)作用,并促進(jìn)纖維化的形成。肝臟中有一類起源于胚胎卵黃囊,只能依靠自我增殖進(jìn)行更新補(bǔ)充的巨噬細(xì)胞,被稱為枯否細(xì)胞(Kupffer cells,KCs)。KCs在肝竇不斷逡巡,能夠吞噬進(jìn)入肝臟中的外界抗原物質(zhì),是保護(hù)肝臟的第一道防線。目前尚不清楚KCs在日本血吸蟲致病過程中的作用。近年來,微小RNA(micro RNA,miRNA)是一個(gè)飽受關(guān)注的研究領(lǐng)域。miRNA是內(nèi)源性19~25個(gè)堿基的小片段非編碼RNA。miRNA可結(jié)合到靶基因3’端非翻譯區(qū)(3’UTR),抑制靶基因m RNA的翻譯或者導(dǎo)致靶基因m RNA降解,從而參與調(diào)控多種疾病的病理過程,如腫瘤發(fā)生、病毒感染、纖維化形成等。本研究首先檢測了小鼠感染日本血吸蟲前后其肝臟KCs的動(dòng)態(tài)變化;在此基礎(chǔ)上,我們篩選日本血吸蟲感染后小鼠肝臟KCs內(nèi)差異表達(dá)的miRNA及m RNA,并采用KEGG軟件,分析這些差異表達(dá)基因的信號通路;最后對其中的一個(gè)差異表達(dá)的miRNA及其靶基因進(jìn)行實(shí)驗(yàn)研究。首先,我們利用密度梯度離心和流式分選相結(jié)合的方法,獲得正常組和感染血吸蟲組小鼠的KCs。在流式分選的過程中,我們發(fā)現(xiàn)日本血吸蟲感染后不同時(shí)間,宿主肝臟內(nèi)的非實(shí)質(zhì)細(xì)胞(non-parenchyma cells,NPCs)存在動(dòng)態(tài)變化。我們判定F4/80~(hi) CD11b~(lo) NPCs細(xì)胞為肝臟KCs;F4/80lo CD11bhi表型細(xì)胞為血液中募集進(jìn)入肝臟的NPCs。在小鼠感染日本血吸蟲的過程中,我們研究了這兩群細(xì)胞的動(dòng)態(tài)變化:對照組的正常小鼠中,肝臟內(nèi)僅有F4/80~(hi) CD11b~(lo)表型的KCs,且數(shù)量穩(wěn)定,在肝臟NPCs中的比例維持在20%左右;而感染日本血吸蟲的小鼠,其F4/80~(hi) CD11b~(lo) KCs的比例開始為20%左右,而隨著血吸蟲病的進(jìn)程,該KCs逐漸減少至少于1%。與此同時(shí),從血液募集的F4/80lo CD11bhi NPCs細(xì)胞群,在感染4周后出現(xiàn),其數(shù)量急劇增加并在感染后期保持穩(wěn)定。肝臟內(nèi)NPCs隨著日本血吸蟲的感染時(shí)間而動(dòng)態(tài)變化,提示我們在日本血吸蟲的不同致病階段中,可能有不同的非實(shí)質(zhì)細(xì)胞參與其中,發(fā)揮相應(yīng)的功能。為了進(jìn)一步探討F4/80~(hi) CD11b~(lo) KCs在日本血吸蟲感染過程逐漸減少的內(nèi)在因素,我們采用miRNA芯片和深度測序的方法,分析日本血吸蟲感染前后宿主KCs miRNA和m RNA表達(dá)譜,并鑒定出差異表達(dá)的miRNA和m RNA。實(shí)驗(yàn)取日本血吸蟲感染后第6周的小鼠以及正常對照組小鼠,分離兩組小鼠肝臟的F4/80~(hi) CD11b~(lo) KCs樣本,提取miRNA和總RNA,分別進(jìn)行miRNA芯片檢測和轉(zhuǎn)錄本測序。miRNA芯片檢測結(jié)果經(jīng)分析顯示:共獲得63個(gè)差異表達(dá)3倍以上的miRNA,其中上調(diào)表達(dá)miRNA 51個(gè),下調(diào)表達(dá)miRNA 12個(gè)。隨機(jī)挑選其中20個(gè)差異表達(dá)的miRNA進(jìn)行實(shí)驗(yàn)驗(yàn)證,經(jīng)實(shí)時(shí)熒光定量PCR(quantitative real time PCR,q RT-PCR)驗(yàn)證,其中17個(gè)和芯片檢測結(jié)果趨勢一致。通過轉(zhuǎn)錄本測序和分析,我們從中篩選到差異表達(dá)2倍以上的基因共1083個(gè),其中上調(diào)表達(dá)494個(gè),下調(diào)表達(dá)589個(gè)。采用KEGG軟件分析,這些差異表達(dá)基因分屬于T細(xì)胞受體信號通路、趨化因子信號通路等10個(gè)信號通路。選取趨化因子相關(guān)通路進(jìn)行q RT-PCR驗(yàn)證,其結(jié)果和轉(zhuǎn)錄本測序結(jié)果趨勢一致。我們進(jìn)一步在感染后4至9周不同時(shí)間點(diǎn)的感染組和對照組樣本中檢測CCL3、CCL4、CCL7等趨化因子,結(jié)果顯示,這些趨化因子在感染后不同時(shí)間點(diǎn)均上調(diào)表達(dá),提示我們?nèi)毡狙x感染后,肝臟內(nèi)NPCs的動(dòng)態(tài)變化可能與趨化因子的增加有關(guān)。在臨床研究中發(fā)現(xiàn),肝癌病人KCs中,CX3CR1的表達(dá)下調(diào)。同時(shí),肝臟內(nèi)KCs中抗凋亡基因Bcl2表達(dá)下調(diào),細(xì)胞有凋亡的趨勢。在四氯化碳(CCL4)誘導(dǎo)小鼠肝纖維化模型的研究中,當(dāng)CX3CR1敲除后,小鼠肝臟的纖維化更加嚴(yán)重。趨化因子受體CX3CR1能夠介導(dǎo)胞外促生存信號傳遞到胞內(nèi),也能夠影響肝臟的纖維化,但是二者之間是否存在關(guān)聯(lián)、其中的具體機(jī)制目前尚不明確。從轉(zhuǎn)錄本測序結(jié)果中我們發(fā)現(xiàn),日本血吸蟲感染后趨化因子受體CX3CR1的表達(dá)與正常小鼠相比有顯著下降。與此同時(shí),我們發(fā)現(xiàn)感染后小鼠肝臟KCs中mi R-297a-5p顯著升高。通過在線靶基因預(yù)測,在CX3CR1基因的3’端非翻譯區(qū)(untranslated region)存在mi R-297a-5p的作用靶點(diǎn)。因此,我們對CX3CR1在日本血吸蟲感染后F4/80~(hi) CD11b~(lo) KCs減少中的作用以及與mi R-297a-5p的靶向關(guān)系開展了進(jìn)一步研究。我們采用q RT-PCR方法,檢測日本血吸蟲感染后不同時(shí)間點(diǎn)感染組和對照組F4/80~(hi) CD11b~(lo) KCs RNA樣本,結(jié)果顯示感染后CX3CR1的表達(dá)呈顯著下調(diào),mi R-297a-5p表達(dá)逐漸增加。此外,感染組和對照組F4/80~(hi) CD11b~(lo) KCs表面CX3CR1流式分析結(jié)果顯示,感染組KCs表面CX3CR1蛋白表達(dá)下調(diào),而對照組CX3CR1蛋白表達(dá)沒有顯著變化。為驗(yàn)證CX3CR1與mi R-297a-5p之間的靶向關(guān)系,我們利用雙熒光報(bào)告系統(tǒng)進(jìn)行了檢測。首先,我們依據(jù)在線預(yù)測的靶向結(jié)合位點(diǎn),合成了野生型CX3CR1 3’UTR結(jié)合位點(diǎn)和突變型結(jié)合位點(diǎn),并插入到帶有熒光基因的報(bào)告載體上。將mi R-297a-5p模擬物(mimics)、野生型和突變型報(bào)告載體分別轉(zhuǎn)染到HEK-293T細(xì)胞中,培養(yǎng)48小時(shí)。經(jīng)檢測,mi R-297a-5p高表達(dá)后,野生型CX3CR1 3’UTR載體報(bào)告基因的熒光表達(dá)會顯著下調(diào)(p0.05),但是突變型CX3CR1 3’UTR載體報(bào)告基因的表達(dá)沒有顯著(p0.05)的變化。為進(jìn)一步研究KCs內(nèi)差異表達(dá)的mi R-297a-5p及其靶基因CX3CR1在日本血吸蟲致病過程中的作用,我們利用巨噬細(xì)胞系,在體外對mi R-297a-5p及CX3CR1進(jìn)行了初步的功能研究。我們利用電轉(zhuǎn)染的方法,將不同濃度的mi R-297a-5p mimics分別轉(zhuǎn)染到KCs細(xì)胞系和RAW264.7細(xì)胞系中。q RT-PCR檢測,mi R-297a-5p表達(dá)量顯著提高,并能夠有效抑制CX3CR1 m RNA的表達(dá)。進(jìn)一步利用流式檢測的方法,對轉(zhuǎn)染細(xì)胞表面CX3CR1蛋白表達(dá)以及凋亡情況進(jìn)行檢測。檢測結(jié)果顯示,細(xì)胞表面CX3CR1蛋白表達(dá)無變化,而且也并未呈現(xiàn)凋亡的現(xiàn)象。在本課題的研究中,我們發(fā)現(xiàn)日本血吸蟲感染后,肝臟內(nèi)NPCs動(dòng)態(tài)變化。其動(dòng)態(tài)變化與日本血吸蟲致病過程有緊密關(guān)聯(lián)。我們篩選出了日本血吸蟲感染后KCs中差異表達(dá)的miRNA和m RNA。對mi R-297a-5p和CX3CR1的靶向關(guān)系及功能進(jìn)行了體外初步研究。本課題為進(jìn)一步研究KCs在日本血吸蟲致病過程中的作用奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R532.21

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