【摘要】：日本血吸蟲主要流行于中國、日本和菲律賓等地,曾在我國肆虐兩千多年之久。六十多年來,我國血吸蟲防治工作取得重大成就,目前我國大部分已處于傳播阻斷的狀況。然而部分地區(qū)尚有數(shù)以萬計晚期血吸蟲病人,且部分省市尚未達到消除血吸蟲的階段,血吸蟲病依然威脅著國人的健康。日本血吸蟲尾蚴經(jīng)皮膚感染,主要危害是由于雌雄成蟲在腸系膜靜脈合抱后產(chǎn)生大量蟲卵,蟲卵在肝臟中沉積并引起劇烈的超敏反應,趨化炎性細胞并形成蟲卵肉芽腫。這種獨特的彌漫性的蟲卵肉芽腫會進行性加重導致肝纖維化,在晚期可引起肝硬化、肝腹水、門脈高壓等不可逆損傷。血吸蟲病嚴重危害人類健康,其多細胞結(jié)構(gòu)和復雜生活史所造成抗原表達的多樣化,導致人們對其引起的復雜免疫病理機制認識太少,這也是該病預防及疫苗研究工作至今仍無突破性進展的重要原因之一。本課題從宿主與病原的相互作用入手,探索日本血吸蟲感染對小鼠免疫環(huán)境變化的影響,尤其關注宿主免疫細胞——Gammadelta(γδ)T在感染前后表型和功能的差異。同時利用抗體阻斷實驗來免疫敲除不同的γδT亞型細胞,確定其對宿主的細胞因子、中性粒細胞和蟲卵肉芽腫纖維化的影響。研究結(jié)果將深化對血吸蟲肝臟病理變化的機制及宿主天然免疫的認識,為進一步開展血吸蟲新型疫苗的設計及優(yōu)化、以及開展免疫治療研究提供新線索和新思路。第一部分日本血吸蟲小鼠模型中γ δ T細胞的表型及功能日本血吸蟲蟲卵沉積于肝臟引起肝臟大量肉芽腫的產(chǎn)生,并引起強烈的免疫病理反應,此進程是長期以來血吸蟲病研究的重點。本研究探討日本血吸蟲小鼠模型中γδT細胞的表型及功能。γδT細胞是介于天然免疫和獲得性免疫之間的一類特殊的T細胞。前人研究血吸蟲模型大多關注于輔助性T(Th)細胞,并認為γδT細胞在肉芽腫形成中無重要影響。但近年來有文獻支持IL-17在血吸蟲感染肝病的進程中發(fā)揮重要作用,且γδT細胞是產(chǎn)生IL-17的主要細胞。本研究著重于闡述γδT細胞在日本血吸蟲小鼠感染模型中,隨著病程發(fā)展的功能變化和可能的作用機制,為進一步闡述IL-17在血吸蟲病中的作用提供依據(jù)。采集血吸蟲感染不同階段小鼠血液和脾臟,利用流式細胞術檢測γδT細胞的表型和功能。同時收集肝臟標本制作冰凍切片,免疫熒光染料染色用于γδT細胞在肉芽腫中的定位分析。同時,感染小鼠收集的脾臟細胞中分離的γδT細胞,進行轉(zhuǎn)錄組芯片分析,初步探究γδT細胞在IL-17通路中可能的作用機制。結(jié)果表明,盡管γδT細胞在淋巴細胞中的百分比無明顯變化(P0.05),但感染后第4周活化的γδT細胞比例增加(P0.05);γδT細胞在血吸蟲病不同階段的肝臟蟲卵肉芽腫中皆存在,且Vγ2亞型也參與其中;Vγ1亞型細胞只產(chǎn)生IFN-γ,Vγ2亞型產(chǎn)生IL17A和IFN-γ,且兩者在感染晚期的細胞功能可能被抑制。γδT細胞可能通過CCL22、CCL1、IL-23a、IL-3、IL-4、MMP9、IL21、CXCL2、CCL2 等因子發(fā)揮功能。第二部分中性粒細胞在日本血吸蟲感染小鼠模型中的表達水平及功能定位在以往研究證實日本血吸蟲感染小鼠模型中,中性粒細胞和巨噬細胞等髓系來源的細胞比例大量增加,這可能與蟲卵肉芽腫的增大及肝纖維化進程有關。在確定γδT細胞與中性粒細胞的招募關系之前,本研究確定中性粒細胞在日本血吸蟲病感染各階段的表達水平,并初步探討其功能定位。首先,采集日本血吸蟲感染各階段小鼠的血液和脾臟,利用多色流式細胞術檢測中性粒細胞在成熟白細胞中的比例;其次,分離感染小鼠脾臟中的中性粒細胞,并與未感染小鼠脾臟來源的T細胞共培養(yǎng),在體外利用羥基熒光素二醋酸鹽琥珀酰亞胺脂(CFSE)檢測中性粒細胞對T細胞增殖的作用;第三,收集血吸蟲感染各階段小鼠的肝臟組織切片,染色中性粒細胞并在激光共聚焦顯微鏡下觀察其在蟲卵肉芽腫中的定位特點;第四,通過尾靜脈注射特異性單克隆抗體抑制中性粒細胞功能,檢測血清IL-17A水平、γδT細胞及其亞型的比例和肝纖維化程度的變化。結(jié)果顯示,血吸蟲感染小鼠模型中,血液和脾臟中的中性粒細胞在白細胞中的比例顯著增加(P0.05);中性粒細胞在體外能抑制T細胞增殖;中性粒細胞在肝臟蟲卵肉芽腫中的定位隨著血吸蟲病的進展而不同,被蟲卵招募至少兩次,并最終全部浸潤肉芽腫;免疫抑制中性粒細胞功能后,Vγ2亞型在γδT細胞中的比例升高(P0.05)。第三部分Vγ2 T細胞在日本血吸蟲感染模型中招募中性粒細胞并促進纖維化γδT細胞能在某些癌癥和自身免疫病模型中通過分泌IL-17招募中性粒細胞來發(fā)揮作用。本研究第二部分已證實日本血吸蟲感染小鼠模型中的中性粒細胞大量增加。該部分假設并驗證“日本血吸蟲感染小鼠模型中γδT細胞能通過分泌IL-17來促進招募中性粒細胞”。首先,運用細胞因子微球檢測(CBA)試劑盒檢測一組感染血吸蟲的小鼠的血清中細胞因子的動態(tài)變化,以此確定IL-17A的基礎水平,為之后的抗體阻斷實驗提供時間點依據(jù)。其次,通過注射抗體分別阻斷γδT細胞及其亞型,檢測血液和脾臟中性粒細胞的比例變化,以及血清中IL-17A的變化。與此同時,收集肝臟切片進行Masson膠原染色和HE染色,并用ELISA試劑盒檢測血清中透明質(zhì)酸(HA)和Ⅲ型前膠原(PCⅢ)的變化,以確定肝臟纖維化程度。另一方面,將γδT細胞和Th細胞過繼轉(zhuǎn)輸至RAG-/-小鼠中,觀察中性粒細胞的在血液和脾臟中的占比變化。結(jié)果表明,隨著血吸蟲病程感染變化,IFN-γ在第5周顯著升高,IL-1β在第6周顯著升高,IL-10從第7周顯著升高,IL-17A在第7周升高,IL-4在第7周升高,G-CSF在第4周開始升高至第7周下降,到第9周再次升高;阻斷總的γδT細胞或單獨阻斷Vγ1T細胞無法顯著減緩中性粒細胞的增加趨勢;單獨阻斷Vγ2T細胞后血清中IL-17A水平下降(P0.05),中性粒細胞在血液和脾臟中的比例降低(P0.05),血清中HA和PCⅢ水平下降(P0.05),蟲卵肉芽腫中的膠原比例下降(P0.05)。Vγ2亞型細胞能分泌IL-17A,促進招募中性粒細胞,可能促進蟲卵肉芽腫的形成,在日本血吸蟲感染初期能加劇纖維化。此外,過繼轉(zhuǎn)輸實驗顯示,在日本血吸蟲感染小鼠模型中,總的γδT細胞和Th細胞都無法顯著提高中性粒細胞的比例,但是Th細胞可能招募嗜酸性粒細胞。第四部分四種常用刺激劑對日本血吸蟲感染小鼠脾臟CD8+ T細胞胞內(nèi)因子及CD62L影響的研究在探討血吸蟲感染小鼠模型體內(nèi)效應性γ δ T細胞和CD8+ T細胞的流式實驗中,本研設計了含有IFN-γ和CD62L的流式配色。但多次實驗發(fā)現(xiàn)均未呈現(xiàn)CD62L的陽性細胞群,而無刺激劑的對照組卻可以檢測到。推測可能是刺激劑導致的CD62L缺失。同時由于本課題第一部分發(fā)現(xiàn)γδT細胞分群特點,改用CD8+T細胞作為研究細胞。為驗證以上假說,本研究主要關注四種常用刺激劑佛波酯(Phorbol-12-myristate-13-acetate,P)、離子霉素(Ionomycin,Ⅰ)、布雷非德菌素 A(BrefeldinA,B)和莫能霉素(Monensin,M)。21 只 C57BL/6 雌性小鼠腹部貼片法感染日本血吸蟲尾蚴,每鼠感染20±2條,建立日本血吸蟲感染小鼠模型。感染后第8周和第12周,取小鼠脾臟制備單細胞懸液,用P+I+M組合體外刺激4 h,利用流式細胞術檢測CD8+ T細胞中分泌IFN-y比例,同時檢測該細胞表面CD62L的表達情況。感染小鼠脾臟單細胞懸液按I組、P組、B組、M、P+I組、P + I + M組、P + I + B組、P + I + M + B組分組,用對應刺激劑體外刺激4 h后,利用細胞因子微球檢測(CBA)法檢測其培養(yǎng)上清中IL-4、IL-17A、IFN-γ、IL-10、IL-1β和集落刺激因子(G-CSF)等細胞因子水平,同時用流式細胞術檢測CD8+T細胞表面CD62L表達情況,計算平均熒光強度(MFI)。結(jié)果表明,日本血吸蟲感染晚期產(chǎn)生IFN-γ的CD8+ T細胞比例減少。細胞經(jīng)P +I刺激之后,CD62L表達下調(diào)。結(jié)論本研究主要探索γδT細胞在血吸蟲感染小鼠模型中對中性粒細胞和肝纖維化的影響,結(jié)論如下:1.日本血吸蟲感染小鼠肝臟蟲卵肉芽腫中存在γδT細胞,Vγ1亞型產(chǎn)生IFN-y,Vγ2亞型同時產(chǎn)生IL-17A和IFN-γ,且兩者在感染晚期的細胞功能可能被抑制。2.中性粒細胞在白細胞中的比例隨著血吸蟲病進展增加,中性粒細胞能抑制T細胞增殖,蟲卵肉芽腫形成過程中發(fā)現(xiàn)中性粒細胞的“二次招募”現(xiàn)象。中性粒細胞是蟲卵肉芽腫的重要細胞。3.Vγ2亞型細胞幫助招募中性粒細胞且可能引起纖維化,單獨阻斷Vγ2亞型細胞后中性粒細胞比例下降,血清IL-17A下降,且肝纖維化程度降低。4.發(fā)現(xiàn)在流式細胞術的實驗設計中應避免同時含有胞內(nèi)因子和CD62L。
[Abstract]:Schistosoma japonicum has been prevalent in China, Japan and the Philippines for more than 2000 years. Great achievements have been made in schistosomiasis control in China over the past 60 years. At present, most of the schistosomiasis cases in China have been blocked. However, there are still tens of thousands of late-stage schistosomiasis cases in some areas, and some provinces and cities have not yet reached it. Schistosomiasis is still a threat to the health of Chinese people during the elimination of schistosomiasis. Percutaneous infection of Schistosoma japonicum cercariae is mainly caused by the large number of eggs produced by adult males and females after their enclosure in the mesenteric vein. The eggs deposit in the liver and cause severe hypersensitivity, chemotaxis inflammatory cells and the formation of egg granuloma. Diffuse egg granuloma can aggravate progressively and lead to liver fibrosis. It can cause irreversible damage such as cirrhosis, ascites, portal hypertension and so on. Schistosomiasis is a serious threat to human health. The diversity of antigen expression caused by its multicellular structure and complex life cycle leads to the understanding of the complex immunopathological mechanism. This is one of the important reasons why there has not been a breakthrough in the prevention and vaccine research of the disease.This study explores the effect of Schistosoma japonicum infection on the immune environment of mice, especially the phenotype and function of host immune cells Gammadelta (gamma delta) T before and after infection, starting from the interaction between host and pathogen. The results will deepen the understanding of the mechanism of liver pathological changes and host innate immunity of Schistosoma japonicum, and further develop a new vaccine for schistosomiasis japonica. Part I The phenotype and function of gamma delta T cells in the mouse model of Schistosoma japonicum. The deposition of Schistosoma japonicum eggs in the liver causes a large number of granulomas in the liver and causes a strong immunopathological reaction. This process has long been the subject of schistosomiasis research. In this study, we investigated the phenotype and function of gamma delta T cells in a mouse model of Schistosoma japonicum. Gamma Delta T cells are a special class of T cells between innate and acquired immunity. It is supported that IL-17 plays an important role in the process of schistosomiasis-infected liver disease and that the main cells producing IL-17 are gamma delta T cells. This study focuses on the functional changes and possible mechanisms of gamma delta T cells in the model of schistosomiasis-infected mice, and further elucidates the role of IL-17 in schistosomiasis. The blood and spleen of mice infected with Schistosoma japonicum at different stages were collected, and the phenotype and function of gamma delta T cells were detected by flow cytometry. At the same time, liver samples were collected for frozen section, and immunofluorescence staining was used to analyze the localization of gamma delta T cells in granulomas. The results showed that although the percentage of gamma delta T cells in lymphocytes did not change significantly (P 0.05), the percentage of activated gamma delta T cells increased at the fourth week after infection (P 0.05); the percentage of gamma delta T cells in egg granulation of liver worms at different stages of schistosomiasis. Vgamma-1 cells only produce IFN-gamma and Vgamma-2 cells produce IL-17A and IFN-gamma, and their cellular functions may be inhibited in the late stage of infection. Gamma Delta T cells may function through CCL22, CCL1, IL-23a, IL-3, IL-4, MMP9, IL-21, CXCL2, CCL2 and other factors. Expression level and functional localization in mice infected with Schistosoma japonicum have been confirmed by previous studies that the proportion of myeloid-derived cells such as neutrophils and macrophages has increased significantly in mice infected with Schistosoma japonicum, which may be related to the enlargement of egg granuloma and the progress of liver fibrosis. In this study, the expression level of neutrophils in various stages of schistosomiasis japonica infection was determined and their functional localization was preliminarily explored. Neutrophils in the viscera were co-cultured with T cells derived from the spleen of uninfected mice, and the effects of neutrophils on T cell proliferation were detected by hydroxyfluorescein diacetate succinimide (CFSE) in vitro. Thirdly, liver sections of mice infected with Schistosoma japonicum were collected, neutrophils were stained and confocal laser scanning was performed. Fourthly, the serum levels of IL-17A, the proportion of gamma delta T cells and their subtypes and the degree of liver fibrosis were detected by tail vein injection of specific monoclonal antibodies to inhibit the function of neutrophils. The proportion of granulocytes in white cells increased significantly (P 0.05); neutrophils inhibited T cell proliferation in vitro; the localization of neutrophils in hepatic egg granulomas varied with the progression of schistosomiasis; the eggs were recruited at least twice and eventually all infiltrated the granulomas; after immunosuppressive neutrophils functioned, the Vgamma 2 subtype was in gamma. The proportion of delta T cells increased (P 0.05). Part III V gamma 2 T cells recruited neutrophils in the Schistosoma japonicum infection model and promoted fibrosis of gamma delta T cells could play a role in some cancer and autoimmune disease models by secreting IL-17 to recruit neutrophils. Part II of this study confirmed that Schistosoma japonicum infected mice. In this part, we hypothesized and validated that in the model of mice infected with Schistosoma japonicum, gamma delta T cells could promote the recruitment of neutrophils by secreting IL-17. Secondly, the ratio of neutrophils in blood and spleen and the change of IL-17A in serum were detected by blocking gamma delta T cells and their subtypes by injection of antibodies. Meanwhile, liver sections were collected for Masson collagen staining and HE staining. The changes of hyaluronic acid (HA) and procollagen type III (PCIII) in serum were detected by ELISA kit to determine the degree of liver fibrosis. On the other hand, gamma delta T cells and Th cells were transfused into RAG - / - mice to observe the changes of neutrophil proportion in blood and spleen. The results showed that IFN - gamma changed with the course of schistosomiasis infection. IL-1 beta increased significantly at the 5th week, IL-10 increased significantly at the 6th week, IL-17A increased significantly at the 7th week, IL-4 increased at the 7th week, G-CSF increased from the 4th week to the 7th week, and increased again at the 9th week. The levels of IL-17A in serum decreased (P 0.05), the proportion of neutrophils in blood and spleen decreased (P 0.05), the levels of HA and PC III in serum decreased (P 0.05), and the proportion of collagen in egg granuloma decreased (P 0.05). In addition, adoptive transfusion experiments showed that the total number of gamma delta T cells and Th cells did not significantly increase the proportion of neutrophils in the mice infected with Schistosoma japonicum, but Th cells may recruit eosinophils. Part IV Four commonly used stimulants were less susceptible to Schistosoma japonicum infection. Study on the effect of CD8+T cell cytokines and CD62L in spleen of mice Flow cytometry was used to study the effectual gamma delta T cells and CD8+T cells in mice infected with Schistosoma japonicum. Flow cytometry with IFN-gamma and CD62L was designed. However, many experiments showed that CD62L positive cells were not found in the spleen of mice infected with Schistosoma japonicum, but the control group without stimulants could. To verify the above hypothesis, this study focused on four commonly used stimulants, Phorbol-12-myristate-13-acetate (P), ionomycin (Ionomycin, I), and Bray. Twenty-one C57BL/6 female mice were infected with Schistosoma japonicum cercariae by abdominal patch method. Each mouse was infected with 20 65 The ratio of IFN-y secreted by CD8+T cells was detected by cytometry, and the expression of CD62L on the surface of CD8+T cells was also detected. The splenic single cell suspensions of infected mice were divided into I group, P group, B group, M, P+I group, P+I+M group, P+I+B group, P+I+M+B group, and P+I+M+B group. After stimulation with corresponding stimulants in vitro for 4 hours, the culture supernatants were detected by cytokine microsphere assay (CBA). Medium levels of IL-4, IL-17A, IFN-gamma, IL-10, IL-1beta and colony-stimulating factor (G-CSF) were measured by flow cytometry, and the expression of CD62L on the surface of CD8+T cells was measured to calculate the mean fluorescence intensity (MFI). The results showed that the proportion of CD8+T cells producing IFN-gamma decreased in the late stage of Schistosoma japonicum infection. Conclusion This study mainly explores the effects of gamma delta T cells on neutrophils and liver fibrosis in mice infected with Schistosoma japonicum. The conclusions are as follows: 1. There are gamma delta T cells in hepatic egg granulomas of mice infected with Schistosoma japonicum, and the subtypes of V gamma 1 produce IFN-y and V gamma 2 produce both IL-17A and IFN-gamma, and both of them have cell functions in the late stage of infection. The proportion of neutrophils in leukocytes increased with the progression of schistosomiasis. Neutrophils inhibited the proliferation of T cells. The phenomenon of "secondary recruitment" of neutrophils was found during the formation of egg granulomas. Neutrophils were important cells in egg granulomas. 3. Vgamma 2 subtypes helped recruit neutrophils. Neutrophils and serum IL-17A were decreased and the degree of liver fibrosis was decreased after blocking Vgamma-2 subtype cells alone. 4. It was found that intracellular factors and CD62L should be avoided in the experimental design of flow cytometry.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R532.21

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