【作者】 张琳；

【导师】 李春香；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2011， 博士

【摘要】 在过去20年里,大量研究证实世界范围内过敏性疾病的发病率逐年增加,其原因不十分清楚。“卫生假说”为儿童在生命早期缺乏微生物的暴露导致今后过敏性疾病的发生进行了解释,由于缺乏微生物刺激导致Th1/Th2平衡紊乱和调节性T细胞功能低下。越来越多研究认为,儿童早期缺乏微生物接触导致的过敏与今后发生哮喘和过敏性疾病的风险密切相关。虽然过敏和哮喘具有遗传倾向性,但基因与环境的相互作用更能解释发病机制。环境因素如感染、接触过敏原以及产前因素如母亲吸烟、孕期营养、产妇精神压力、分娩方式和新生儿期应用抗生素等均会影响小儿今后过敏性疾病和支气管哮喘的发生。通过基因与环境的相互作用、表观遗传学以及分子生物学深入研究,力图解开假定风险与过敏性疾病和支气管哮喘发生的关联。有关哮喘和过敏性紫癜的流行病学资料、危险因素调查以及病理机制有待进一步解释,了解这些机理对减少哮喘发生风险及寻找切实有效的药物干预微生态生境,调整免疫功能对过敏性疾病的预防是十分必要的。第一部分:肠道微生态学变化、CD4+T细胞亚群及相应转录因子在儿童支气管哮喘病理机制中的调控作用目的:探讨肠道微生态环境变化以及免疫网络结构中CD4+T亚群及相应细胞因子和上游转录因子在支气管哮喘病理机制中的相互调控作用。方法:采用流式细胞术(Flow cytometry,FCM)对47例支气管哮喘患儿和32例健康对照组儿童外周血单个核细胞(Peripheral blood mononuclear cell , PBMC )测定CD3+CD8-INF-γ+ IL-4- ( Th1 )、CD3+CD8-INF-γ- IL-4(+Th2)、CD4+CD25+T细胞(Treg)和CD3+CD8-IL-17+( Th17)细胞百分比;采用酶联免疫吸附试验( Enzyme linked immunosorbent assay,ELISA)测定血浆中细胞因子INF-γ、IL-4、TGF-β1和IL-17水平;T-bet是调控Th1细胞分化、GATA3是调控Th2细胞分化、Foxp3是调控调节性T细胞分化、ROR-γt是Th17分化的关键性转录因子,采用逆转录聚合酶链反应( Reverse transcription-polymerase chain reaction ,RT-PCR)检测外周血单个核细胞内T-bet、GATA3、Foxp3和ROR-γt mRNA的表达;采用细菌16S rDNA方法测定粪便中双歧杆菌、乳酸杆菌、大肠杆菌和肠球菌含量。结果:支气管哮喘患儿在急性发作期时发生肠道菌群失调,表现在双歧杆菌和乳酸杆菌为代表的厌氧菌数量下降,大肠杆菌和肠球菌为代表的需氧菌数量变化不大,双大肠杆菌歧杆菌/(Bifidobacteria/ escherichia coli, B/E)值下降。随着治疗和症状改善,恢复期时双歧杆菌数量上升较其他细菌为快,但整体菌群结构并未扶正。对于免疫学指标的观察发现,支气管哮喘患儿在急性发作期时外周血PBMC中Th2和Th17细胞百分比以及血浆中Th2和Th17细胞因子IL-4和IL-17浓度均高于健康对照组;Th1和Treg细胞百分比以及血浆中Th1和Treg细胞因子INF-γ和TGF-β1浓度均低于健康对照组,出现Th1/Th2和Treg/Th17失衡。缓解期Th2和Th17细胞百分比及血浆相应细胞因子浓度随疾病的回复而降低,但仍高于健康对照组,仍存在免疫紊乱。支气管哮喘患儿Th1细胞与血浆中INF-γ浓度和外周血PBMC中T-bet表达呈正相关;Th2细胞与血浆中IL-4浓度和外周血PBMC中GATA3表达呈正相关;Treg细胞与血浆中TGF-β1浓度和外周血PBMC中Foxp3表达呈正相关;Th17细胞与血浆中IL-17浓度和外周血PBMC中ROR-γt表达呈正相关,表明Th1/Th2和Treg/Th17主要受其特异性转录因子调控。肠道菌群中B/E值与Th1/Th2和Tregs/Th17之间均呈正相关关系。结论:Th1/Th2细胞分化失衡在支气管哮喘发病机制中发挥重要作用,除了Th2细胞免疫应答活跃外,调节性T细胞以及Th17细胞也参与了在支气管哮喘的发病过程中,表明除了经典的Th1/Th2免疫失衡外,还存在Treg/Th17免疫失调。肠道共生菌刺激机体免疫系统的发育,在支气管哮喘中通过下调Th2应答,纠正Th1/Th2失衡。肠道菌群结构和功能的完整性对维系CD4+CD25+T细胞功能发重要作用,支气管哮喘患儿肠道菌群失调损伤了CD4+CD25+T细胞的免疫抑制功能,导致免疫耐受被打破是支气管哮喘病理过程的重要机制。其研究结论为今后药品的开发和应用,并在早期及时恢复肠道微生态平衡,阻断异常免疫的某个环节,防治支气管哮喘提供了重要理论依据。第二部分:肠粘膜屏障功能、CD4+T细胞亚群及相应转录因子在儿童过敏性紫癜病理机制中的调控作用目的:探讨肠粘膜屏障功能变化及其免疫网络中CD4+T亚群及相应细胞因子和特异性转录因子在过敏性紫癜病理机制中的相互调控作用。方法:采用流氏细胞术测定41例过敏性紫癜患儿和30例健康儿童外周血单个核细胞CD3+CD8-INF-γ+ IL-4(-Th1), CD3+CD8-INF-γ- IL-4+(Th2),CD4+CD25+T细胞(Treg)和CD3+CD8-IL-17+(Th17)细胞百分比;采用酶联免疫吸附试验(ELISA)测定血浆中INF-γ、IL-4、TGF-β1、IL-9和IL- 17细胞因子水平;T-bet是调控Th1细胞分化,GATA3是调控Th2细胞分化,Foxp3是调控调节性T细胞分化,ROR-γt是控制Th17分化的特异性转录因子,由逆转录聚合酶链反应(RT-PCR)检测外周血PBMC内T-bet、GATA3、Foxp3和ROR-γt mRNA表达;采用细菌16S rDNA方法测定粪便中双歧杆菌、乳酸杆菌、大肠杆菌和肠球菌含量。采用液相色谱仪测定尿中乳果糖(lactulose, L)与甘露醇(mannito, M)水平,计算L/M值评价肠粘膜通透性。结果:过敏性紫癜患儿在急性期即发生肠道菌群失调,表现为肠道中双歧杆菌和乳酸杆菌下降,大肠杆菌和肠球菌减少不明显,以乳酸杆菌数量下降为显著。治疗后随着临床症状的改善,恢复期时双歧杆菌数量上升较乳酸杆菌为快,肠道仍处于微生态紊乱状态,并且肠道中乳酸杆菌数量和B/E值和尿L/M值呈负相关。在过敏性紫癜急性期时,外周血PBMC中Th2和Th17细胞亚群百分比以及血浆中IL-4、IL-17水平和特异性转录因子GATA3、ROR-γt mRNA表达均高于健康对照组;Th1和Treg细胞百分比以及血浆中INF-γ、TGF-β1水平和特异性转录因子T-bet、Foxp3 mRNA表达均低于健康对照组,出现Th1/Th2和Treg/Th17免疫失衡。恢复期时Th2和Th17细胞百分比及IL-4、IL-17水平和特异性转录因子GATA3和ROR-γt表达有所下降,但仍存在Th1/Th2、Treg/Th17免疫失衡。过敏性紫癜患儿血浆中INF-γ与外周血PBMC中Th1细胞百分比、T-bet表达呈正相关;IL-4与外周血PBMC中Th2细胞百分比、GATA3表达呈正相关;TGF-β1与外周血PBMC中Treg细胞百分比、Foxp3表达呈正相关;IL-17与外周血PBMC中Th17细胞百分比、ROR-γt表达呈正相关。肠道菌群中B/E值和乳酸杆菌数量分别与Th1/Th2和Tregs/Th17呈正相关,与尿L/M比值呈负相关。结论:过敏性紫癜患儿在急性期出现肠道微生态失衡,表现在肠道菌群B/E值下降,乳酸杆菌数量下降尤为显著,肠粘膜通透性下降,肠粘膜屏障功能遭到损坏,肠道共生菌所诱导的免疫耐受机制被打破,表现在外周血PBMC中CD4+CD25+T细胞百分比降低及其血浆TGF-β1水平降低,转录因子Foxp3表达下降,对以往经典的Th1/Th2细胞免疫失衡的发病理论进行了修订,认为过敏性紫癜的发病机制除了有Th1/Th2免疫失衡外,也存在Treg/Th17的免疫失调,并参与了疾病的病理过程。肠道乳酸杆菌和双歧杆菌在恢复机体免疫自稳状态,诱导免疫耐受方面发挥重要作用。研究结论从微生态学和免疫学角度阐明了过敏性紫癜的发病机制,探讨了肠道共生菌、免疫细胞、细胞因子以及转录因子在疾病发生发展中的调控作用,为今后药品的开发不仅限于调整宿主的免疫功能,更要放眼于改善肠道微生态环境,为过敏性紫癜的临床治疗提供前期的理论依据。第三部分参芪健脾方对肠道菌群失调大鼠肠粘膜屏障功能修复及免疫功能调整作用机制的实验研究目的:前两部分研究已表明儿童过敏性疾病如支气管哮喘和过敏性紫癜的病理过程均发生肠道微生态失调和免疫调控机能失衡,故寻求改善微生态生境,调节免疫功能的药物,早期阻断疾病的恶性循环,防治过敏性疾病十分重要。本实验探讨参芪健脾方对肠道菌群失调大鼠修复肠粘膜屏障功能、抑制肠道细菌移位和调整免疫功能的作用机制研究,对开发中药方剂防治过敏性疾病具有重要临床价值。方法:采用细菌培养法动态测定大鼠肠道菌群结构变化及肠系膜淋巴结、肝脏、脾脏和结肠组织的移位细菌量;应用光镜和电子显微镜观察大鼠肠粘膜组织超微结构变化;采用逆转录聚合酶链反应(RT-PCR)动态测定大鼠肠系膜淋巴结、肝脏、脾脏和肠粘膜组织中Toll样受体mRNA转录水平。结果:肠道菌群失调模型建立,大鼠肠道中双歧杆菌、乳酸杆菌和肠杆菌数量均较对照组下降。肠粘膜组织受损,表现在肠绒毛、肠上皮细胞以及紧密连接的受损。大鼠体内Toll样受体mRNA转录水平早期受抑制,肠系膜淋巴结、肠粘膜、肝脏和脾脏组织出现肠道细菌移位情况。大肠杆菌攻击可加重肠道菌群失调和肠粘膜损伤程度,上调Toll样受体mRNA转录水平,加剧细菌移位程度。参芪健脾方和乳酸杆菌可减轻由肠道菌群失调引起的Toll样受体mRNA转录受抑及细菌移位程度,扶正肠菌群结构,修复损伤的肠粘膜屏障功能。结论:阐明了肠道菌群、肠粘膜屏障功能和肠道细菌移位以及免疫功能之间的互为因果,相互影响的关系。肠道菌群失调改变了肠道微生态生境,损伤了肠粘膜上皮细胞结构,促发肠道细菌移位,致使Toll样受体mRNA转录受抑。参芪健脾方在维护机体微生态平衡、修复肠粘膜屏障功能和抑制肠道细菌移位方面相同于乳酸杆菌制剂,参芪健脾方可调整机体免疫功能,尤其是天然免疫的模式识别受体,对于过敏性疾病的早期防治具有临床价值。更多还原

【Abstract】 Many studies have confirmed increases in the incidence and prevalence of allergic diseases over the past 2 decades in the worldwide, but much remains unknown. These observations support the hygiene hypothesis, i.e. the lack of microbial exposure during infancy or early childhood leading to allergic diseases. Moreover, the lack of microbial stimulation also results in the imbalance of Th1/Th2 and hypofunction of regulatory T cells. There is growing evidence that early-life allergy is significantly related to the development of asthma and other allergic diseases. Although genetic predisposition is clearly evident, gene-by-environment interaction probably explains much of the international variation in prevalence rates for asthma and other allergic diseases. Environmental factors such as infections and exposure to endotoxins,some prenatal risk factors, including maternal smoking, diet and nutrition, stress, use of antibiotics and mode of delivery may also affect the early development of asthma and other allergic diseases. It is likely that detailed studies of gene-by-environment interactions, molecular biology and epigenetics will eventually untangle the inconsistencies among the many putative exposures and outcomes. Much of the epidemiology, many of environment risk factors and pathophysiological mechanisms in asthma and Henoch-Schonlein purpura remain to be adequately explained. A better understanding of these mechanisms may reduce the risk factors for asthma and find effective drugs to regulate gut microecology and immune function, which will eventually lead to opportunities for primary prevention of allergic diseases. Part one: Alteration of gut microecology and regulation of the CD4+T cell subsets and their cytokines and master transcription factors in immune networks in bronchial asthma pathogenesisObjective: To study alteration of gut microecology and mutual regulation of the CD4+ T cell subsets and their cytokines and master transcription factors in immune networks in bronchial asthma pathogenesis.Methods: Forty-seven children with asthma and thirty healthy children as control group were enrolled into this study. Flow cytometrie (FCM) analysis was performed to detect the percentage of CD3+CD8-INF-γ+ IL-4-(Th1),CD3+CD8-INF-γ-IL-4+(Th2),CD4+CD25+T cell(Treg)and CD3+CD8-IL-17+(Th17)cell; Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of INF-γ,IL -4,TGF-β1 and IL- 17 in plasma ; The specific master transcription factors for Th1, Th2, Treg and Th17 cells, T-bet, GATA3, Foxp3 and ROR-γt, are respectively involved in their differentiation. Reverse transcription-polymerase chain reaction(RT-PCR) was used to analyze the mRNA expression of T-bet, GATA3, Foxp3 and ROR-γt in peripheral blood mononuclear cell (PBMC).16SrDNA fluorescent quantitative PCR was applied in determining the content of bifidobacterium, lactobacillus,escherichia coli and enterococcus in feces.Results: The amount of anaerobes represented by the bifidobacterium and lactobacillus declined. Howere, the amount of aerobes represented by escherichia coli and enterococcus did not change obviously, Bifidobacteria / Escherichia coli (B/E) which indicated a balance of intestinal flora declined. The results showed the intestinal dysbacteriosis occured in children with acute stage of asthma. With treatment and the improvement of symptoms, the amount of bifidobacteria increased faster than other bacteria, but the overall flora structure did not restore to its normal levels in recovery stage. Based on the immunological indexes in children with acute stage of asthma, the percentage of Th2 and Th17 cells and the concentrations of their related cytokines in plasma increased, and the percentage of Th1 and Treg cells and the concentrations of their related cytokines decreased as compared to that in the control group, indicating that there were immune imbalances of Treg/Th17 and Th1/Th2. Though the percentage of Th2 and Th17 cell and the concentrations of cytokines in plasma decreased in remission of asthma, they were still higher than that in healthy control group, and there was still the imbalance of Treg/Th17 and Th1/Th2. Analysis of the correlation between the level of cytokines in blood plasma and the expression of specific master transcription factors in PBMC showed that positive correlations between INF-γand T-bet, IL-4 and GATA3, TGF-β1 and Foxp3, and IL-17 and ROR-γt in children with bronchial asthma, indicating that the balances of Th1/Th2 and Treg/Th17 was mainly regulated by their master transcription factors. Th1/Th2 ratio and Tregs/Th17 ratio had positive correlation with B / E ratio.Conclusions: The imbalance of Th1/Th2 cells is critical in the pathogenetic mechanisms of bronchial asthma. Besides its importance for Th2 cell development, Treg cell and Th17 cell play significant roles in the process. Thus, besides the imbalance of Th1/Th2 cells, the imbalance of Treg/Th17 cells also involved in the pathogenetic mechanisms of bronchial asthma. Commensal intestinal bacteria are crucial in providing immune stimulation for normal immune system development, and can correct the imbalance of Th1/Th2 by downregulating Th2 responses. The integrity of intestinal flora structure and their function are important to maintain the function of CD4+CD25+ Treg cell. Intestinal dysbacteriosis in children with bronchial asthma compromises the ability of CD4+CD25+Treg cell to suppress responses, the process leading to the disturbance of immune tolerance which is important in asthma pathogenesis. Our study have provided an important theoretical basis for future development and application of medicines, and prevention and treatment of bronchial asthma through timely restoring the balance of gut microecology and abrogating any component element of abnormal immune responses at early stages. Part two: Alteration of gut microecology and regulation of the CD4+ T cell subsets and their cytokines and master transcription factors in immune networks in pathogenesis of Henoch-Schonle in purpura in childrenObjective: To study alteration of gut microecology and mutual regulation of the CD4+ T cell subsets and their cytokines and master transcription factors in immune networks in pathogenesis of Henoch-Schonlein purpura in children.Methods: Forty-one children with Henoch-Schonlein purpura and thirty healthy children as control were enrolled into this study. Flow cytometrie (FCM) analysis was performed to detect the percentage of CD3+CD8-INF-γ+ IL-4-(Th1), CD3+CD8-INF-γ- IL-4+(Th2), CD4+CD25+T cell(Treg)and CD3+CD8-IL-17+(Th17)cell; enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of INF-γ, IL -4,TGF-β1, IL-9 and IL- 17 in plasma ;the master transcription factors for Th1, Th2, Treg and Th17 cells, T-bet, GATA3, Foxp3 and RORγt, are respectively involved in their differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of T-bet, GATA3, Foxp3 and ROR-γt in peripheral blood mononuclear cell (PBMC). 16S rDNA fluorescent quantitative PCR was applied in determining the bacterial content of bifidobacterium, lactobacillus, escherichia coli and enterococcus in feces. Urine samples were collected before and after the test solution administrated ( lactulose, L and mannitol, M) and analyzed by high performance liquid chromatography (HPLC) and the intestinal permeability can be evaluated by L/M ratio.Results: The amount of the bifidobacterium and lactobacillus declined, especially lactobacillus. However, the amount of escherichia coli and enterococcus did not change obviously. The results showed that intestinal dysbacteriosis occured in children with HSP during acute stage. With clinical treatment and the improvement of symptoms, the amount of bifidobacterium and lactobacillus gradually increased, and the amount of bifidobacterium increased faster than that of lactobacillus, but there was still intestinal dysbacteriosis during the recovery stage of HSP. Moreover, the amount of lactobacillus had negative correlation with B/E and L/M ratio. Based on the other indexes during the acute stage of HSP, the percentage of Th2 and Th17 cells in PBMC, the levels of IL-4 and IL-17 in plasma, the expression of GATA3 and ROR-γt in PBMC increased when compared with that in the control group. The percentage of Th1 and Treg cells, the levels of INF-γand TGF-β1, the expression of T-bet and Foxp3 decreased as compared to that in the control group. The results indicated that there were imbalances of Treg/Th17 and Th1/Th2 during the acute stage of HSP. Though the percentage of Th2 and Th17 cells, the levels of their cytokines in plasma and the expression of their master transcription factors increased during the recovery stage of HSP, there were still the imbalance of Treg/Th17 and Th1/Th2. Analysis of the correlation between the expression of master transcription factors in PBMC, the percentage of cells in PBMC and the levels of cytokines in plasma in children with HSP showed that Th1 and T-bet had positive correlations with INF-γ; Th2 and GATA3 had positive correlations with IL-4; Treg and Foxp3 had positive correlations with TGF-β1; Th17 and ROR-γt had positive correlations with IL-17. Similar to B/E ratio, the amount of the lactobacillus also had positive correlations with Th1/Th2 ratio and Tregs/Th17 ratio and negative correlation with L/M ratio.Conclusion: B/E ratio decreased, especially for the amount of lactobacillus, intestinal permeability increased, and intestinal barrier function was damaged, indicating that the intestinal dysbacteriosis occurred in children with HSP in acute stage. The decrease of the percentage of CD4+CD25+T cell, the level of TGF-β1 in plasma and the expression of Foxp3 in PBMC showed that immune tolerance induced by commensal intestinal bacteria was reversed. The finding was a supplement to Th1/Th2 paradigm .In our study, besides Th1/Th2 paradigm, the imbalance of Treg/Th17cells was involved in the pathogenesis of HSP. Bifidobacterium and lactobacillus in gut played significant roles in restoration of the immune homeostasis and tolerance induction. From the ecological and immunological perspective, our study clarified pathogenesis of allergic purpura and investigated the regulation of commensal intestinal bacteria, immune cells, cytokines and transcription factors in the development of the disease, which provided theoretical basis for future development of medicines and clinical treatment of Henoch-Schonlein purpura not only through regulating the host immune function, but also through improving gut microecology.Part three : The mechanisms of ShenQiJianPi decoction in restoring gut mucosa barrier function and modulating immune function in SD rats with intestinal dysbacteriosis.Object: Based on the studies above, we demonstrated that intestinal dysbacteriosis and immune function disorders were involved in pathogenesis of allergic diseases such as bronchial asthma and allergic purpura in children. Therefore, it is very important to develop new traditional medicines which can improve gut microecology, regulate immune function and break vicious cycle at early stage of allergic diseases. This study investigated the mechanisms of ShenQiJianPi decoction in restoring the gut mucosa barrier function, inhibiting bacterial translocation phenomenon and modulating of immune function in SD rats with intestinal dysbacteriosis, which provided theoretical basis for future development of traditional chinese medicines to treat allergic diseases.Methods: The dynamic changes of intestinal flora and the amount of translocated bacteria in mesenteric lymph nodes, liver, spleen and colon in SD rats were observed by bacterial culture after using antibiotic; bowel tissue electronmicroscopic and opticalmicroscopic changes in SD rats were performed; the reverse transcription polymerase chain reaction (RT-PCR)was used for determining Toll-like receptor mRNA expression of intestinal tissues, mesenteric lymph nodes, liver and spleen from SD rats.Results: The amount of the bifidobacterium, lactobacillus and escherichia coli decreased as compared to that in the control group, indicating that the intestinal dysbacteriosis occured in SD rats with intestinal dysbacteriosis. Concerning indexes of measuring intestinal barrier function, intestinal epithelial cell, the intestinal villus and intestinal tight junction were damaged, suggesting that intestinal barrier integrity loss occured. The expression of Toll-like receptor mRNA was inhabited in the early of intestinal dysbacteriosis, which leading to bacterial translocation from gastrointestinal tract to mesenteric lymph nodes, liver and spleen. Escherichia coli pathogenic attack may increase the degree of intestinal dysbacteriosis and the damage gut barrier function, while the upregulation of Toll-like receptor mRNA expression may increase the degree of bacterial translocation. ShenQiJianPi decoction and lactobacillus intervention could be applied to reduce the degree of bacterial translocation, lessen the inhibition of Toll-like receptor of mRNA expression, restore the structure of intestinal flora and lessen the damage gut barrier function.Conclusion: The correlations among the structure of intestinal flora, gut mucosa barrier function, bacterial translocation phenomenon and immune function have been clarified. Intestinal dysbacteriosis can induce the alteration of gut microecology, damage of intestinal epithelial cells, bacterial translocation phenomenon and inhabition the expression of Toll-like receptor mRNA. Early intervention of ShenQiJianPi decoction provided the protective effect on maintaining the balance in microecosystem, restoring gut barrier function, inhibiting bacterial translocation phenomenon and modulating the immune function , especially on modulating host pattern recognition receptor in innate immunity, which are critical in the prevention of allergic disease.更多还原