日本血吸虫感染宿主滤泡辅助性T细胞变化及功能的研究

发布时间：2018-05-12 12:02

本文选题：日本血吸虫 + 滤泡辅助性T细胞　；参考：《中国疾病预防控制中心》2016年博士论文

【摘要】：全球约有2亿人感染血吸虫,是严重的公共卫生问题。我国流行的是日本血吸虫病。日本血吸虫感染引起宿主的免疫应答机制复杂,人们对其认识不足,这是限制血吸虫病防治工作及日本血吸虫疫苗研究的瓶颈。国内外很多学者关注于Th1、Th2、Th17及Treg等细胞在日本血吸虫感染宿主中的作用,而关于滤泡辅助性T细胞(T follicular helper cells, Tfh)的研究则不多,其在日本血吸虫感染中的作用机制尚不清楚。Tfh细胞属于CD4+T细胞的一个亚群,促进生发中心(germinal center, GC)的生成及生发中心B细胞产生抗体。最近的研究发现,Tfh细胞表面的ICOS分子能促进日本血吸虫感染小鼠中肝脏肉芽肿的形成,而Tfh细胞在日本血吸虫感染中其他方面的免疫作用及其机制还未阐明。本研究针对日本血吸虫感染患者,探索Tfh细胞表型及功能变化；进一步以日本血吸虫感染的C57BL/6小鼠为模型,分析Tfh细胞在感染小鼠的表型及功能；基于Tfh细胞在日本血吸虫感染小鼠和血吸虫病患者的研究结果,以日本血吸虫感染小鼠为模型,采用Agilent小鼠芯片分析日本血吸虫感染前后Tfh细胞差异表达的基因,筛选相关的免疫调控分子,为研究日本血吸虫感染小鼠的免疫应答机制提供科学依据和分子基础；此外,观察日本血吸虫感染小鼠经吡喹酮治疗后对Tfh细胞的影响。本研究期望通过研究Tfh细胞在日本血吸虫感染过程的作用,加深对宿主免疫反应机制的理解,将为血吸虫病的免疫与疫苗的进一步研究提供科学依据和新思路。1.日本血吸虫病患者Tfh细胞及相关分子的研究Tfh细胞属于CD4+T细胞的一个亚群,促进生发中心的生成及生发中心B细胞产生抗体,本研究对Tfh细胞在血吸虫病患者中的特征进行了探讨。本研究通过流式细胞术检测和分析了湖南省血吸虫病流行区的急性日本血吸虫病患者(12例)、慢性日本血吸虫病患者(11例)和健康对照者(10例)外周血Tfh细胞的比例及其表面分子的表达水平,并检测了B细胞亚群的分布情况。酶联免疫吸附试验(enzyme linked immunosorbent assay, ELIS A)检测患者血清中IL-21水平及血吸虫可溶性虫卵抗原(soluble egg antigen, SEA)和可溶性成虫抗原(soluble adult worm antigen, SWA)特异性抗体水平,并探讨了Tfh细胞与日本血吸虫特异性抗体之间的相关性。结果表明,急性血吸虫病患者Tfh细胞表面的PD-1分子(7.42%±3.15%)表达比健康对照组(3.96%±1.05%)明显增高(P0.01)；慢性血吸虫病患者Tfh细胞比例(23.67%±3.12%)比健康对照组(18.31%±6.34%)增高(P0.01)、其表面分子ICOS(12.44%±2.84%)和PD-1(5.38%±1.42%)表达也比健康对照组(ICOS:9.51%±2.46%;PD-1:3.96%±1.05%)明显增高(P0.05),而且慢性血吸虫病患者的PD-1+CXCR5+CD4+Tfh细胞与血清中IL-21(rs=0.782,P=0.004)、SEA特异性的IgG(rs=0.688,P=0.028).IgGl(rs=0.709,P=0.015)及IgG4(rs=0.527,P=0.024)呈明显的正相关。本研究首次在日本血吸虫病患者中进行Tfh细胞的研究,Tfh细胞可通过表面分子及分泌的细胞因子参与免疫应答,PD-1+CXCR5+CD4+ Tfh细胞可能在日本血吸虫特异性抗体的产生中发挥重要作用,有助于进一步理解血吸虫病免疫应答机制。2.Tfh细胞在日本血吸虫感染小鼠中的表型及功能研究本研究以日本血吸虫感染C57BL/6纯系小鼠为模型,通过免疫荧光等技术对Tfh细胞进行定位观察。在感染的不同时间(感染后3w、5w、8w和13w)应用流式细胞术检测Tfh细胞比例及其表型特征；分析日本血吸虫可溶性虫卵抗原对淋巴细胞及Tfh细胞增殖能力的影响以及生发中心B细胞的变化。首先采用免疫荧光和流式细胞术发现脾脏Tfh细胞表达CD4+ CXCR5+PD-1+;通过动物体内实验进行动态观察Tfh细胞在CD4+T细胞的比例,发现Tfh细胞的比例随着日本血吸虫感染时间的延长逐渐增高,8w到达高峰,随后逐渐降低；日本血吸虫SEA和SWA分别免疫小鼠后,日本血吸虫SEA和SWA这两种抗原均可引起Tfh细胞比例(4.868%±0.650%,4.380%±0.444%)增加(P0.01)；SEA和SWA分别在体外刺激脾脏淋巴细胞,发现Tfh细胞的比例(12.268%±0.672%,11.532%±1.967%)也明显增加(P0.05)。体外实验研究表明,日本血吸虫感染小鼠脾脏淋巴细胞的增殖(0.573±0.021)比未感染小鼠脾脏淋巴细胞的增殖(1.407±0.147)能力降低(P0.05),但感染组Tfh细胞的增殖(1.090±0.157)和未感染组(1.41±0.155)相比,增殖能力未见明显下降,并且感染组Tfh分泌IL-21(0.240±0.034)的能力明显高于正常组(0.143±0.015)(P0.05)。生发中心B细胞被活化,其比例与Tfh细胞的变化趋势一致。Tfh细胞在日本血吸虫感染中的作用及其分子机制还有待进一步阐明。3.日本血吸虫感染小鼠Tfh细胞差异分子筛选基于前两部分的研究,我们发现Tfh细胞在日本血吸虫感染小鼠和血吸虫病患者的外周血或脾脏中明显增高,且慢性日本血吸虫病患者PD-1+CXCR5+CD4+Tfh细胞与SEA特异的IgG抗体呈明显正相关,但其确切机制尚不清楚。本研究以日本血吸虫感染小鼠为模型,通过小鼠基因表达谱芯片筛选日本血吸虫感染组和正常组小鼠脾脏的Tfh细胞相关的免疫调控分子。小鼠感染日本血吸虫后8周,与正常小鼠一起取脾脏,磁珠分选分离CD4+T细胞,再利用流式细胞仪分选PD-1+CXCR5+CD4+Tfh细胞。抽提分选细胞的总RNA进行小鼠表达谱分析,再通过生物信息学分析筛选感染前后PD-1+CXCR5+CD4+Tfh细胞的差异表达基因,并对筛选出的差异基因进行GO和KEGG注释等分析,同时挑选部分差异表达的基因利用荧光定量PCR方法进行验证。比较表明,感染组与未感染正常组的PD-1+CXCR5+CD4+Tfh,发现差异表达基因有1506个,其中上调的基因642个,下调的基因864个。这些基因的主要功能包括粒细胞迁移、炎症反应中白细胞的迁移、粒细胞趋化、内皮细胞迁移的正调节、急性炎症反应、细胞因子分泌的正调节和细胞因子受体活性等。挑选了15个基因,利用荧光定量PCR进行验证,其中14个基因与芯片筛选的结果一致。进一步比较感染组小鼠PD-1+CXCR5+CD4+Tfh细胞与PD-1+CXCR5+CD4+ non-Tfh细胞的差异表达基因,差异表达基因有710个,其中上调的基因406个,下调的基因304个。这些基因的功能包括细胞迁移的正调控、免疫应答的正调控、MAPK级联的正调节、B细胞受体信号途径、通过MHCⅡ类抗原加工和呈递抗原的外源肽、细胞因子受体活性等。挑选12个基因,利用荧光定量PCR进行验证,其中10个与芯片筛选的结果一致。本研究所筛选的日本血吸虫感染组和正常组的PD-1+CXCR5+CD4+Tfh细胞及感染组PD-1+CXCR5+CD4+Tfh细胞与PD-1-CXCR5+CD4+non-Tfh细胞之间的差异基因将为进一步研究Tfh细胞在日本血吸虫感染中的免疫作用及其机制提供科学依据和分子基础。4.日本血吸虫感染小鼠吡喹酮治疗后Tfh细胞及相关分子研究目前治疗日本血吸虫病唯一有效化学药物吡喹酮影响宿主的免疫应答,我们前期研究结果表明Tfh细胞参与日本血吸虫感染宿主的免疫应答,吡喹酮是否通过Tfh而影响宿主的免疫应答?因此本研究观察日本血吸虫感染小鼠经吡喹酮治疗对Tfh细胞的影响。C57BL/6雌性小鼠经日本血吸虫感染后,随机分为日本血吸虫感染毗喹酮治疗组(治疗组)、日本血吸虫感染未治疗组(未治疗组),与正常未感染日本血吸虫对照组(对照组)进行比较。治疗组的小鼠于感染后6周给予200 mg/(kg·d)吡喹酮灌胃治疗,每天一次,连续治疗3 d。治疗4周后解剖各组小鼠,观察小鼠肝脏和脾脏病变情况,流式细胞术分析小鼠外周血和脾脏Tfh细胞及其表面分子ICOS和PD-1的表达情况。结果表明,吡喹酮治疗后,日本血吸虫感染小鼠肝脏、脾脏组织的病变明显改善,检获的成虫及虫卵的数量明显减少,减虫率和肝脏减卵率分别为84.06%和69.11%(P均0.01)。治疗组小鼠外周血和脾脏Tfh表面的ICOS(0.7%～1.1%和1.8%～6.8%)的表达比未治疗组(1.3%～3.2%和4.1%～7.0%)显著降低,可作为吡喹酮疗效评价的参考指标之一。结论本课题探索日本血吸虫感染对宿主Tfh细胞的影响,主要取得以下结果：1.急性和慢性血吸虫病患者的Tfh细胞的重要表面分子发生显著变化,急性血吸虫病患者Tfh细胞比例未见增高,但Tfh细胞表面的PD-1分子表达增高；而慢性血吸虫病患者Tfh细胞比例增高,其表面分子ICOS和PD-1表达也增高。慢性血吸虫病患者的PD-1+CXCR5+ CD4+ Tfh细胞与血清中IL-21、SEA特异的IgG、IgGl及IgG4抗体呈明显的正相关。提示Tfh细胞在日本血吸虫感染的不同阶段所发挥免疫功能随着其表面重要分子的变化而有所不同。2.采用免疫荧光技术对小鼠脾脏中的Tfh细胞进行定位,结合流式细胞术分析本研究中小鼠Tfh细胞的表面标记为PD-1+ CXCR5+ CD4+。日本血吸虫感染引起脾脏淋巴细胞增殖能力下降,而Tfh细胞的增殖能力未见明显下降,且Tfh细胞分泌IL-21明显增加,促使脾脏淋巴滤泡生发中心B细胞被活化。动态分析表明,生发中心B细胞与Tfh细胞的变化趋势是一致的,即感染初期增高,8w时最高,随后降低。3.采用Agilent小鼠芯片分析日本血吸虫感染前后PD-1+ CXCR5+ CD4+ Tfh细胞及感染组PD-1+CXCR5+CD4+Tfh细胞与PD-1-CXCR5+CD4+non-Tfh细胞的差异基因,筛选出一批免疫功能相关分子,为Tfh细胞的功能深入研究提供了分子基础。4.吡喹酮治疗后,日本血吸虫感染小鼠肝、脾组织病变明显减轻,寄生的成虫及虫卵数量明显减少。外周血和脾脏Tfh细胞ICOS的表达比未治疗组显著降低,有可能作为吡喹酮疗效评价的参考指标之一。
[Abstract]:About 200 million people in the world are infected with Schistosoma japonicum, which is a serious public health problem. The epidemic of schistosomiasis in China is Japanese schistosomiasis. The immune response mechanism of the host is complicated and the understanding of it is insufficient. This is the bottleneck of the prevention and control of schistosomiasis and the research of the vaccine of Schistosoma japonicum. Many scholars at home and abroad pay attention to Th1, The role of Th2, Th17 and Treg cells in the host of Schistosoma japonicum infection, while the research on follicular auxiliary T cells (T follicular helper cells, Tfh) is not much. The mechanism of its role in Schistosoma japonicum infection is not clear that.Tfh cells belong to a subgroup of CD4+T cells and promote the generation of the germinal center (germinal). Recent studies have found that ICOS molecules on the surface of Tfh cells can promote the formation of liver granuloma in mice infected with Schistosoma japonicum, and other aspects of the immune function and mechanism of Tfh cells in Schistosoma japonicum infection have not been elucidated. This study aims at the exploration of Tfh cells in patients with Schistosoma japonicum infection. The phenotype and function change of the C57BL/6 mice infected by Schistosoma japonicum were further analyzed. The phenotype and function of Tfh cells in infected mice were analyzed. Based on the results of Tfh cells in mice infected with Schistosoma japonicum and schistosomiasis, the mice infected with Schistosoma japonicum were modeled and the Japanese blood sucking was analyzed by the Agilent mouse chip. The differentially expressed genes of Tfh cells before and after the infection and screening related immunoregulatory molecules provide a scientific basis and molecular basis for the study of the immune response mechanism of mice infected with Schistosoma japonicum. In addition, the effects of praziquantel on Tfh cells in mice infected with Schistosoma japonicum were observed. The purpose of this study was to study the Tfh cells in the day. The role of the infection process of Schistosoma japonicum will deepen the understanding of the host immune response mechanism and provide a scientific basis and new ideas for the further study of the immunization and vaccine of schistosomiasis.1.. The study of Tfh cells and related molecules of schistosomiasis japonica patients, Tfh cells belong to a subgroup of CD4+T cells, promoting the generation and development of the germinal center. This study explored the characteristics of Tfh cells in patients with schistosomiasis in this study. This study detected and analyzed the acute Japanese schistosomiasis (12 cases), chronic schistosomiasis (11 cases) and healthy controls (10 cases) of Tfh cells in the peripheral blood of the Schistosomiasis Area in Hunan province. The expression level of the proportion and its surface molecules and the distribution of B cell subsets were detected. The enzyme linked immunosorbent assay (enzyme linked immunosorbent assay, ELIS A) was used to detect the level of IL-21 in the sera and the soluble egg antigen of Schistosoma (soluble egg antigen, SEA) and soluble adult antigen The correlation between Tfh cells and the specific antibody of Schistosoma japonicum was investigated. The results showed that the expression of PD-1 (7.42% + 3.15%) on the surface of Tfh cells in patients with acute schistosomiasis was significantly higher than that in the healthy control group (3.96% + 1.05%) (P0.01), and the proportion of Tfh cells (23.67% + 3.12%) in the patients with chronic schistosomiasis was higher than that of the healthy ones. The group (18.31% + 6.34%) increased (P0.01), its surface molecules ICOS (12.44% + 2.84%) and PD-1 (5.38% + 1.42%) were also significantly higher than those of the healthy control group (ICOS:9.51% + 2.46%; PD-1:3.96% + 1.05%), and the PD-1+CXCR5+CD4+Tfh cells in the chronic schistosomiasis patients and the serum IL-21 (rs=0.782, P=0.004), SEA specific IgG 028).IgGl (rs=0.709, P=0.015) and IgG4 (rs=0.527, P=0.024) have obvious positive correlation. This study is the first time to study Tfh cells in Japanese schistosomiasis patients. Tfh cells can participate in the immune response through surface molecules and secreted cytokines, and PD-1+CXCR5+CD4+ Tfh cells may play a role in the production of specific antibodies in Schistosoma japonicum. The important role is to further understand the phenotype and function of the immune response mechanism of schistosomiasis.2.Tfh cells in mice infected with Schistosoma japonicum. This study takes the C57BL/6 mice infected with Schistosoma japonicum as a model, and localize the Tfh cells by immunofluorescence techniques. At different times of infection (after infection 3W, 5W, 8W) And 13W) use flow cytometry to detect the proportion and phenotypic characteristics of Tfh cells, analyze the effect of soluble egg antigen of Schistosoma japonicum on the proliferation of lymphocyte and Tfh cells and the changes of B cells in the germinal center. First, the expression of CD4+ CXCR5+PD-1+ in the spleen Tfh cells was detected by immunofluorescence and flow cytometry; the expression of CD4+ CXCR5+PD-1+ in the spleen was detected by the immunofluorescence and flow cytometry; The proportion of Tfh cells in CD4+T cells was observed dynamically, and the proportion of Tfh cells increased gradually with the prolongation of the time of infection of Schistosoma japonicum, the peak of 8W reached the peak and then decreased gradually. After immunization with SEA and SWA of Schistosoma japonicum, the two antigens of Schistosoma japonicum, SEA and SWA, could cause the proportion of Tfh cells (4.868% + 0.650%). 4.380% + 0.444%) increased (P0.01); SEA and SWA stimulated spleen lymphocytes in vitro, and found that the proportion of Tfh cells (12.268% + 0.672%, 11.532% + 1.967%) also increased significantly (P0.05). In vitro, the proliferation of splenic lymphocytes in mice infected with Schistosoma japonicum (0.573 + 0.021) was compared to the proliferation of spleen lymphocytes in uninfected mice. (1.407 + 0.147) decreased (P0.05), but the proliferation of Tfh cells in infected group (1.090 + 0.157) and uninfected group (1.41 + 0.155) were not significantly decreased, and the ability of Tfh to secrete IL-21 (0.240 + 0.034) in the infection group was significantly higher than that of the normal group (0.143 + 0.015) (P0.05). The B cells in the germinal center were activated, and the proportion of the cells and the changes of Tfh cells were changed. The role of trend consistent.Tfh cells in Schistosoma japonicum infection and its molecular mechanism still need to be further elucidated in the first two parts of the screening of Tfh cell differentials in.3. infected mice. We found that Tfh cells were significantly higher in peripheral blood or spleen of Schistosoma japonicum infected mice and schistosomiasis patients. The PD-1+CXCR5+CD4+Tfh cells of the chronic Japanese schistosomiasis patients are positively correlated with the specific IgG antibody of SEA, but the exact mechanism is not clear. This study uses the mice infected with Schistosoma japonicum as a model to screen the immune regulation related to the Tfh cells of the spleen of the Schistosoma japonicum infection group and the normal group of mice by the mouse gene expression chip. 8 weeks after infection of Schistosoma japonicum, mice were taken spleen with normal mice, magnetic beads were selected to separate CD4+T cells, and then PD-1+CXCR5+CD4+Tfh cells were selected by flow cytometry. The total RNA of the selected cells was extracted and the expression profiles of the mice were analyzed. Then the difference table of PD-1+CXCR5+CD4+Tfh cells before and after infection was screened by bioinformatics analysis. GO and KEGG annotations were performed on the selected genes, and the selected genes were selected by the fluorescence quantitative PCR method. The results showed that there were 1506 differentially expressed genes in the infected and uninfected PD-1+CXCR5+CD4+Tfh groups, including 642 up regulated genes and 864 down-regulated genes. The main functions of these genes include granulocyte migration, leukocyte migration in the inflammatory response, granulocyte chemotaxis, positive regulation of endothelial cell migration, acute inflammatory response, positive regulation of cytokine secretion and cytokine receptor activity. 15 genes were selected and verified by fluorescein quantitative PCR, of which 14 genes were screened with chips. The difference of expression genes between PD-1+CXCR5+CD4+Tfh cells and PD-1+CXCR5+CD4+ non-Tfh cells in infected mice was further compared. There were 710 differentially expressed genes, including 406 up-regulated genes and 304 down-regulated genes. The functions of these genes include positive regulation of cell migration, positive regulation of immune response, and positive modulation of the MAPK cascade. The B cell receptor signaling pathway, through the MHC class II antigen processing and presenting antigen exogenous peptide, cytokine receptor activity, selected 12 genes and verified by fluorescence quantitative PCR, 10 of them were consistent with the results of chip screening. The PD-1+CXCR5+CD4+Tfh cells of Schistosoma japonicum infection group and the normal group screened by this study The differential gene between PD-1+CXCR5+CD4+Tfh cells and PD-1-CXCR5+CD4+non-Tfh cells in the infected group will provide a scientific basis and molecular basis for further study of the immune function of Tfh cells in the infection of Schistosoma japonicum and its molecular basis,.4., Tfh cells and related molecules after the treatment of praziquantel in mice infected with Schistosoma japonicum, and the present Study on the treatment of Japanese blood The only effective drug, praziquantel, affects the immune response of the host. Our previous study showed that Tfh cells were involved in the immune response of the host infected with Schistosoma japonicum. Whether praziquantel could affect the host's immune response through Tfh? Therefore, this study observed the effects of praziquantel on Tfh cells in mice infected with Schistosoma japonicum. After infected with Schistosoma japonicum, the female mice of.C57BL/6 were randomly divided into the treatment group (treated group), the untreated group of Schistosoma japonicum (untreated group), compared with the normal control group (control group). The mice in the treatment group were given 200 mg/ (kg. D) praziquantel for 6 weeks after infection. Treatment, once a day, after 3 d. treatment for 4 weeks, the mice were dissected and the pathological changes of liver and spleen were observed. Flow cytometry was used to analyze the expression of Tfh cells and their surface molecules ICOS and PD-1 in the peripheral blood and spleen of mice. The results showed that after the treatment of praziquantel, the liver and spleen tissues of mice infected with Japanese Schistosoma japonicum were infected. The number of infected adults and eggs decreased significantly, the rate of worm reduction and the rate of liver reduction were 84.06% and 69.11% (P 0.01) respectively. The expression of ICOS (0.7% ~ 1.1% and 1.8% to 6.8%) on the surface of peripheral blood and spleen on the Tfh of the treatment group was significantly lower than that in the untreated group (1.3% to 3.2% and 4.1% ~ 7%), which could be used as a reference for the evaluation of the effect of praziquantel. The main results are as follows: 1. significant changes in the surface molecules of Tfh cells in patients with acute and chronic schistosomiasis have been significantly changed, and the proportion of Tfh cells in acute schistosomiasis patients has not increased, but the expression of PD-1 molecules on the surface of Tfh cells is higher. The proportion of Tfh cells in the patients with chronic schistosomiasis was increased and the expression of its surface molecules ICOS and PD-1 increased. The PD-1+CXCR5+ CD4+ Tfh cells of the patients with chronic schistosomiasis were positively correlated with the IL-21, SEA specific IgG, IgGl and IgG4 antibodies in the serum, suggesting that the immune function of the Tfh cells in different stages of the infection of Schistosoma japonicum is associated with its immune function The changes in surface important molecules are different and different.2. uses immunofluorescence technique to locate Tfh cells in the spleen of mice. In combination with flow cytometry, the surface marker of mouse Tfh cells in this study is PD-1+ CXCR5+ CD4+., and the proliferation ability of spleen lymphocyte in the spleen is decreased, but the proliferation ability of Tfh cells is not found. Obviously decreased, and the secretion of IL-21 in Tfh cells increased significantly, prompting the activation of B cells in the germinal center of the lymphoid follicle. The dynamic analysis showed that the trend of B cells in the germinal center and Tfh cells was the same, that is, the initial increase of the infection and the highest 8W, and then the.3. using Agilent mouse chip to analyze PD-1+ CXCR5+ before and after the infection of Schistosoma japonicum. CD4+ Tfh cells and infected group PD-1+CXCR5+CD4+Tfh cells and PD-1-CXCR5+CD4+non-Tfh cell differentially screened a number of immune function related molecules, providing a molecular basis for the function of Tfh cells to provide a molecular basis for the study of the molecular basis of the treatment of praziquantel, the liver of mice infected with Schistosoma japonicum, the pathological changes of spleen tissue, parasitic adults and eggs. The expression of ICOS in peripheral blood and spleen Tfh cells was significantly lower than that in the untreated group. It may be used as a reference index for the evaluation of praziquantel efficacy.

【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】：2016
【分类号】：R532.21

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