日本血吸虫感染小鼠γδT细胞对中性粒细胞及肝纤维化作用的研究
发布时间：2018-09-11 17:36
【摘要】：日本血吸虫主要流行于中国、日本和菲律宾等地,曾在我国肆虐两千多年之久。六十多年来,我国血吸虫防治工作取得重大成就,目前我国大部分已处于传播阻断的状况。然而部分地区尚有数以万计晚期血吸虫病人,且部分省市尚未达到消除血吸虫的阶段,血吸虫病依然威胁着国人的健康。日本血吸虫尾蚴经皮肤感染,主要危害是由于雌雄成虫在肠系膜静脉合抱后产生大量虫卵,虫卵在肝脏中沉积并引起剧烈的超敏反应,趋化炎性细胞并形成虫卵肉芽肿。这种独特的弥漫性的虫卵肉芽肿会进行性加重导致肝纤维化,在晚期可引起肝硬化、肝腹水、门脉高压等不可逆损伤。血吸虫病严重危害人类健康,其多细胞结构和复杂生活史所造成抗原表达的多样化,导致人们对其引起的复杂免疫病理机制认识太少,这也是该病预防及疫苗研究工作至今仍无突破性进展的重要原因之一。本课题从宿主与病原的相互作用入手,探索日本血吸虫感染对小鼠免疫环境变化的影响,尤其关注宿主免疫细胞——Gammadelta(γδ)T在感染前后表型和功能的差异。同时利用抗体阻断实验来免疫敲除不同的γδT亚型细胞,确定其对宿主的细胞因子、中性粒细胞和虫卵肉芽肿纤维化的影响。研究结果将深化对血吸虫肝脏病理变化的机制及宿主天然免疫的认识,为进一步开展血吸虫新型疫苗的设计及优化、以及开展免疫治疗研究提供新线索和新思路。第一部分日本血吸虫小鼠模型中γ δ T细胞的表型及功能日本血吸虫虫卵沉积于肝脏引起肝脏大量肉芽肿的产生,并引起强烈的免疫病理反应,此进程是长期以来血吸虫病研究的重点。本研究探讨日本血吸虫小鼠模型中γδT细胞的表型及功能。γδT细胞是介于天然免疫和获得性免疫之间的一类特殊的T细胞。前人研究血吸虫模型大多关注于辅助性T(Th)细胞,并认为γδT细胞在肉芽肿形成中无重要影响。但近年来有文献支持IL-17在血吸虫感染肝病的进程中发挥重要作用,且γδT细胞是产生IL-17的主要细胞。本研究着重于阐述γδT细胞在日本血吸虫小鼠感染模型中,随着病程发展的功能变化和可能的作用机制,为进一步阐述IL-17在血吸虫病中的作用提供依据。采集血吸虫感染不同阶段小鼠血液和脾脏,利用流式细胞术检测γδT细胞的表型和功能。同时收集肝脏标本制作冰冻切片,免疫荧光染料染色用于γδT细胞在肉芽肿中的定位分析。同时,感染小鼠收集的脾脏细胞中分离的γδT细胞,进行转录组芯片分析,初步探究γδT细胞在IL-17通路中可能的作用机制。结果表明,尽管γδT细胞在淋巴细胞中的百分比无明显变化(P0.05),但感染后第4周活化的γδT细胞比例增加(P0.05);γδT细胞在血吸虫病不同阶段的肝脏虫卵肉芽肿中皆存在,且Vγ2亚型也参与其中;Vγ1亚型细胞只产生IFN-γ,Vγ2亚型产生IL17A和IFN-γ,且两者在感染晚期的细胞功能可能被抑制。γδT细胞可能通过CCL22、CCL1、IL-23a、IL-3、IL-4、MMP9、IL21、CXCL2、CCL2 等因子发挥功能。第二部分中性粒细胞在日本血吸虫感染小鼠模型中的表达水平及功能定位在以往研究证实日本血吸虫感染小鼠模型中,中性粒细胞和巨噬细胞等髓系来源的细胞比例大量增加,这可能与虫卵肉芽肿的增大及肝纤维化进程有关。在确定γδT细胞与中性粒细胞的招募关系之前,本研究确定中性粒细胞在日本血吸虫病感染各阶段的表达水平,并初步探讨其功能定位。首先,采集日本血吸虫感染各阶段小鼠的血液和脾脏,利用多色流式细胞术检测中性粒细胞在成熟白细胞中的比例;其次,分离感染小鼠脾脏中的中性粒细胞,并与未感染小鼠脾脏来源的T细胞共培养,在体外利用羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)检测中性粒细胞对T细胞增殖的作用;第三,收集血吸虫感染各阶段小鼠的肝脏组织切片,染色中性粒细胞并在激光共聚焦显微镜下观察其在虫卵肉芽肿中的定位特点;第四,通过尾静脉注射特异性单克隆抗体抑制中性粒细胞功能,检测血清IL-17A水平、γδT细胞及其亚型的比例和肝纤维化程度的变化。结果显示,血吸虫感染小鼠模型中,血液和脾脏中的中性粒细胞在白细胞中的比例显著增加(P0.05);中性粒细胞在体外能抑制T细胞增殖;中性粒细胞在肝脏虫卵肉芽肿中的定位随着血吸虫病的进展而不同,被虫卵招募至少两次,并最终全部浸润肉芽肿;免疫抑制中性粒细胞功能后,Vγ2亚型在γδT细胞中的比例升高(P0.05)。第三部分Vγ2 T细胞在日本血吸虫感染模型中招募中性粒细胞并促进纤维化γδT细胞能在某些癌症和自身免疫病模型中通过分泌IL-17招募中性粒细胞来发挥作用。本研究第二部分已证实日本血吸虫感染小鼠模型中的中性粒细胞大量增加。该部分假设并验证“日本血吸虫感染小鼠模型中γδT细胞能通过分泌IL-17来促进招募中性粒细胞”。首先,运用细胞因子微球检测(CBA)试剂盒检测一组感染血吸虫的小鼠的血清中细胞因子的动态变化,以此确定IL-17A的基础水平,为之后的抗体阻断实验提供时间点依据。其次,通过注射抗体分别阻断γδT细胞及其亚型,检测血液和脾脏中性粒细胞的比例变化,以及血清中IL-17A的变化。与此同时,收集肝脏切片进行Masson胶原染色和HE染色,并用ELISA试剂盒检测血清中透明质酸(HA)和Ⅲ型前胶原(PCⅢ)的变化,以确定肝脏纤维化程度。另一方面,将γδT细胞和Th细胞过继转输至RAG-/-小鼠中,观察中性粒细胞的在血液和脾脏中的占比变化。结果表明,随着血吸虫病程感染变化,IFN-γ在第5周显著升高,IL-1β在第6周显著升高,IL-10从第7周显著升高,IL-17A在第7周升高,IL-4在第7周升高,G-CSF在第4周开始升高至第7周下降,到第9周再次升高;阻断总的γδT细胞或单独阻断Vγ1T细胞无法显著减缓中性粒细胞的增加趋势;单独阻断Vγ2T细胞后血清中IL-17A水平下降(P0.05),中性粒细胞在血液和脾脏中的比例降低(P0.05),血清中HA和PCⅢ水平下降(P0.05),虫卵肉芽肿中的胶原比例下降(P0.05)。Vγ2亚型细胞能分泌IL-17A,促进招募中性粒细胞,可能促进虫卵肉芽肿的形成,在日本血吸虫感染初期能加剧纤维化。此外,过继转输实验显示,在日本血吸虫感染小鼠模型中,总的γδT细胞和Th细胞都无法显著提高中性粒细胞的比例,但是Th细胞可能招募嗜酸性粒细胞。第四部分四种常用刺激剂对日本血吸虫感染小鼠脾脏CD8+ T细胞胞内因子及CD62L影响的研究在探讨血吸虫感染小鼠模型体内效应性γ δ T细胞和CD8+ T细胞的流式实验中,本研设计了含有IFN-γ和CD62L的流式配色。但多次实验发现均未呈现CD62L的阳性细胞群,而无刺激剂的对照组却可以检测到。推测可能是刺激剂导致的CD62L缺失。同时由于本课题第一部分发现γδT细胞分群特点,改用CD8+T细胞作为研究细胞。为验证以上假说,本研究主要关注四种常用刺激剂佛波酯(Phorbol-12-myristate-13-acetate,P)、离子霉素(Ionomycin,Ⅰ)、布雷非德菌素 A(BrefeldinA,B)和莫能霉素(Monensin,M)。21 只 C57BL/6 雌性小鼠腹部贴片法感染日本血吸虫尾蚴,每鼠感染20±2条,建立日本血吸虫感染小鼠模型。感染后第8周和第12周,取小鼠脾脏制备单细胞悬液,用P+I+M组合体外刺激4 h,利用流式细胞术检测CD8+ T细胞中分泌IFN-y比例,同时检测该细胞表面CD62L的表达情况。感染小鼠脾脏单细胞悬液按I组、P组、B组、M、P+I组、P + I + M组、P + I + B组、P + I + M + B组分组,用对应刺激剂体外刺激4 h后,利用细胞因子微球检测(CBA)法检测其培养上清中IL-4、IL-17A、IFN-γ、IL-10、IL-1β和集落刺激因子(G-CSF)等细胞因子水平,同时用流式细胞术检测CD8+T细胞表面CD62L表达情况,计算平均荧光强度(MFI)。结果表明,日本血吸虫感染晚期产生IFN-γ的CD8+ T细胞比例减少。细胞经P +I刺激之后,CD62L表达下调。结论本研究主要探索γδT细胞在血吸虫感染小鼠模型中对中性粒细胞和肝纤维化的影响,结论如下:1.日本血吸虫感染小鼠肝脏虫卵肉芽肿中存在γδT细胞,Vγ1亚型产生IFN-y,Vγ2亚型同时产生IL-17A和IFN-γ,且两者在感染晚期的细胞功能可能被抑制。2.中性粒细胞在白细胞中的比例随着血吸虫病进展增加,中性粒细胞能抑制T细胞增殖,虫卵肉芽肿形成过程中发现中性粒细胞的“二次招募”现象。中性粒细胞是虫卵肉芽肿的重要细胞。3.Vγ2亚型细胞帮助招募中性粒细胞且可能引起纤维化,单独阻断Vγ2亚型细胞后中性粒细胞比例下降,血清IL-17A下降,且肝纤维化程度降低。4.发现在流式细胞术的实验设计中应避免同时含有胞内因子和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.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R532.21
[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.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R532.21
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