广州管圆线虫感染小鼠脑损伤导致免疫抑制及其机制的研究
发布时间：2018-07-13 10:21
【摘要】：广州管圆线虫是引起嗜酸粒细胞增多性脑膜炎和脑膜脑炎的重要病原体。人是广州管圆线虫的非适宜宿主,多因生食或半生食含有广州管圆线虫Ⅲ期幼虫的中间宿主而感染。感染期幼虫可穿过肠壁进入血液循环系统,随血流在体内移行,具有嗜神经性,多侵犯中枢神经系统。该病的特征为脑脊液中嗜酸性粒细胞升高,病变除了大脑和脑膜还可波及小脑、脑干、脊髓或眼球,主要的病变有脑充血、出血、脑组织机械性损伤及肉芽肿反应。近些年来由于广州管圆线虫中间宿主福寿螺和褐云玛瑙螺在我国南方地区大量繁殖,加上人们饮食上嗜好生猛鲜活与猎奇,导致广州管圆线虫病相继在各地暴发或散发流行,已被列入新发感染性疾病。目前临床上常用阿苯达唑驱虫,但是虫体死亡时崩解释放大量的抗原引起的炎症反应会导致神经系统症状的加重。因此,深入了解广州管圆线虫感染脑损伤的免疫应答机制对于广州管圆线虫病的防治具有重要意义。研究表明中枢神经系统和免疫系统以复杂的方式双向相互作用。在理想的情况下,应激条件下的炎症和抗炎反应是平衡的,有利于创伤愈合和遏制病原体,同时防止过高的炎症反应或严重的免疫抑制。然而,在没有全身性炎症的情况下,脑损伤局部炎性细胞因子扩散触发的抗炎反应可能是有害的,因为这样会下调机体的防御机制,使机体容易受到感染。有研究发现,中枢神经系统损伤对免疫功能产生深远的影响,如中风,创伤性脑损伤或脊髓损伤后的患者均表现出免疫功能受损,包括外周血淋巴细胞数量减少,脾脏和胸腺萎缩,T细胞活性受损等。进一步研究发现,急性中枢神经系统受损患者循环单核细胞主要组织相容性复合体II类分子表达下调,体外内毒素刺激单核细胞,其产生促炎性细胞因子的能力大大降低。这种免疫抑制的临床表现为在脑损伤后的不久高发全身性感染,特别是肺炎和尿路感染,而感染会阻碍神经的恢复、增加患者死亡率。这些研究结果提示,脑损伤会导致外周免疫抑制。广州管圆线虫的幼虫侵入人体后移行至大脑,可造成脑组织机械损伤,占位并释放代谢产物损伤中枢神经系统。基于脑部损伤与外周免疫抑制的密切联系,我们提出如下假说：广州管圆线虫幼虫侵入大脑损伤中枢神经系统后,导致大脑局部炎性细胞因子扩散刺激抗炎反应引起外周免疫抑制,增加机体的继发感染的机会。为了验证上述假说,在小鼠广州管圆线虫感染模型上,我们首先观察了广州管圆线虫感染后免疫器官胸腺和脾脏的变化,同时对外周淋巴细胞亚群数量和功能的变化进行了评估；其次,我们从细胞凋亡和细胞发育受阻两方面探讨了广州管圆线虫感染后外周淋巴细胞数量下降的原因；再次,我们研究了广州管圆线虫感染后脑组织炎性细胞因子、抗炎细胞因子和趋化因子的表达,观察中枢神经系统与外周免疫系统联系的下丘脑-垂体-肾上腺(HPA)轴的变化；最后,我们证实在广州管圆线虫感染中枢神经系统损伤后,糖皮质激素促进了外周的免疫抑制。本研究获得如下主要结果：1.广州管圆线虫感染诱导免疫器官萎缩和淋巴细胞数量减少感染后21天小鼠的胸腺和脾脏较对照组小鼠明显萎缩,且单个核细胞数也减少,提示广州管圆线虫感染可影响小鼠的免疫器官。为了探讨感染后胸腺和脾脏萎缩的原因,我们用流式细胞仪检测了小鼠脾脏和外周血细胞亚群的变化。检测结果表明,感染后脾脏和外周血中B细胞、T细胞、CD4+T细胞、CD8+T细胞、Thl细胞及NK细胞的数量较对照组显著减少。2.广州管圆线虫感染降低细胞免疫和体液免疫功能为了证实在广州管圆线虫感染后,淋巴细胞的免疫功能也受感染的影响,我们进一步研究了T淋巴细胞增殖功能、分泌细胞因子和B细胞产生抗体的能力。结果显示,感染后小鼠T细胞的增殖能力较对照组小鼠降低；采用有丝分裂原刺激外周全血细胞,其培养上清用ELISA检测的结果显示,广州管圆线虫感染诱导低水平的IFN-γ、TNF-α和高水平的IL-4;用非特异性抗原OVA免疫广州管圆线虫感染小鼠和对照组小鼠,检测OVA-IgGl抗体表达水平。检测结果显示,感染后小鼠体内OVA-IgGl抗体水平较对照小鼠降低,提示广州管圆线虫感染降低了小鼠B细胞产生抗体的能力。为了进一步证实这些结果,我们观察了小鼠肺部的病理和采集小鼠的血液做菌血培养。结果显示,感染后小鼠肺部有大量炎性细胞浸润,表现为肺炎。同样血液在血琼脂平板上培养显示感染后出现菌血症,表明广州管圆线虫感染诱导了小鼠免疫抑制。3.广州管圆线虫感染增强NK细胞的杀伤功能 因为广州管圆线虫感染降低了细胞免疫和体液免疫功能,导致小鼠的感染风险增加,所以我们进一步评估了广州管圆线虫感染对NK细胞功能的影响。我们在加或不加外源性IL-12的条件下对NK细胞刺激培养24 h, ELISA检测培养上清中IFN-γ和TNF-α的表达水平。结果表明,IL-12处理的感染小鼠NK细胞较对照小鼠产生更高水平的IFN-γ。无论是否存在外源性IL-12,广州管圆线虫感染小鼠和对照小鼠均只能诱导NK细胞产生低水平的TNF-β。进一步我们评估了感染对NK细胞杀伤活性的影响,结果显示,感染后的NK细胞较对照组小鼠有更强的杀伤功能。这些结果提示,广州管圆线虫感染虽然降低了NK细胞的数量,但是增强了NK细胞的杀伤功能。4.广州管圆线虫感染后B细胞和T细胞的减少不是由于凋亡所导致,但NK细胞在感染后出现大量的凋亡感染后B和T细胞数量急剧降低,为了进一步明确细胞数量下降是否由凋亡所致,我们用流式检测了这些细胞表面7AAD-AnnexinV+的比例。结果发现,与对照组相比,感染后细胞表面7AAD" Annexin V+比例没有增加。Caspase 3作为凋亡最终的执行者,是检测凋亡的一个重要指标。为了进一步证实这个结果,我们用磁珠分选的方法分选出B220-细胞和CD3-细胞,用estern blot的方法检钡Cleaved Caspase 3的表达,结果表明感染后没有检测至Cleaved Caspase 3表达的增加,提示广州管圆线虫感染后B细胞和T细胞数量的下降不是由于凋亡所导致的。同样地,为了明确NK细胞数量下降是否由凋亡所致,我们用流式检测了NK细胞表面7AAD" Annexin V+的比例及用磁珠分选的方法分选出DX5+细胞,用estern blot的方法检狈Cleaved Caspase 3的表达。结果发现,与对照组相比,感染后NK细胞表面7AAD- Annexin V+和Cleaved Caspase 3的表达增加,提示广州管圆线虫感染导致NK数量的降低与NK细胞发生的凋亡有关。5.广州管圆线虫感染抑制骨髓中B细胞的发生和损伤T细胞在胸腺的发育广州管圆线虫感染后B细胞数量的降低不是由于细胞凋亡所引起的,我们推测在B细胞的发育的过程中,是否所有或部分发育B细胞亚群有损失。为了验证这一猜想,我们检测了骨髓发育中B细胞的比例。结果所示,感染后骨髓发育中B细胞的比例较对照组降低,进一步,我们分析了骨髓发育中B细胞的两个亚群：pro-/pre-B细胞和不成熟B细胞。结果表明：感染后骨髓中pro-/pre-B细胞占发育中B细胞的比例较对照组显著降低；而感染后骨髓中不成熟B细胞占发育中B细胞比例较对照组上升,提示广州管圆线虫感染导致骨髓发育中B细胞下降,其中受主要影响的是pro-/pre-B细胞。我们知道pro-/pre-B细胞在Ig H链和L链基因重排后,骨髓中的发育中B细胞进入到不成熟B细胞阶段,然后迁移到外周进入到过渡期B细胞。进一步我们检测了脾脏中发育中B细胞和成熟B细胞的比例,结果显示感染后21天,成熟B细胞降低。同时,脾脏发育中B细胞的比例较对照组显著降低。进一步,我们分析了发育中B细胞的3个阶段亚群：TR1、TR2和TR3亚群。结果表明,随着感染时间的增加,TR1占发育中B细胞的比例逐步降低。TR2和TR3占发育中B细胞的比例随着感染时间的增加而增加。提示广州管圆线虫感染导致脾脏发育中B细胞下降,其中受主要影响的是TR1细胞亚群。综合以上结果,提示由于广州管圆线虫感染影响B细胞的发生,从而导致B细胞输出下降。广州管圆线虫感染后T细胞数量的降低同样不是由细胞凋亡所引起,并且在感染后胸腺出现严重的萎缩,我们推测是否T细胞在胸腺的发育过程受阻。为此我们用流式细胞仪检测了胸腺T细胞亚群,结果显示感染后CD4+CD8+T细胞的比例较对照组降低,与之相反的是随着感染时间的增加,CD4-CD8-T细胞、CD4+CD8-T细胞和CD4-CD8+T细胞的比例较对照组显著增高。进一步我们观察了胸腺各T细胞亚群的数量,结果显示,感染后CD4CD8T细胞和CD4"CD8+T细胞的数量较对照组没有差异；但是感染后CD4+CD8+T细胞的数量较对照组显著降低。为了进一步明确其细胞数量下降是否由凋亡所致,我们用流式检测了CD4+CD8+T细胞表面7AAD" Annexin V+的表达。结果表明,与对照组相比,感染后CD4+CD8+T细胞表面7AAD-Annexin V+比例逐步增加。综合以上结果,提示广州管圆线虫感染后胸腺细胞数量减少和外周T细胞数量的减少与CD4+CD8+T细胞凋亡后减少有关。6.广州管圆线虫感染导致的脑损伤引起淋巴细胞数量减少和下丘脑-垂体-肾上腺轴的活化广州管圆线虫幼虫移行进入脑内,侵犯神经系统,破坏脑组毛织。为了明确广州管圆线虫感染导致的淋巴细胞数量的降低是否由于脑损伤导致,我们采用阿苯达唑杀虫实验观察感染后杀虫对外周淋巴细胞亚群的影响。结果表明,杀虫处理组在感染后21天胸腺和脾脏大小较正常对照组没有显著的差异。感染后杀虫脾脏淋巴细胞亚群B细胞、T细胞、CD4+T细胞、CD8+T细胞和NK细胞的比例较正常对照组也没有显著差异,提示广州管圆线虫感染后外周出现免疫抑制发生在幼虫富集于脑组织导致CNS损伤之后,也即是感染导致的脑损伤诱导了外周的免疫抑制。为了明确广州管圆线虫脑损伤后的外周免疫抑制是否与下丘脑-垂体-肾上腺轴的活化从而引起糖皮质激素分泌增加有关,我们检测了下丘脑-垂体-肾上腺轴活化的指标。结果发现,感染后血浆中皮质酮含量的表达较对照组相比显著增加；小鼠海马、下丘脑室旁核、垂体和肾上腺c-fos mRNA的表达水平显著增加；下丘脑室旁核中促肾上腺皮质激素释放激素和肾上腺中肾上腺酪氨酸羟化酶的mRNA的表达水平较对照组表达水平显著增加。以上结果提示,广州管圆线虫感染会导致下丘脑-垂体-肾上腺轴的活化。7.广州管圆线虫感染后阻断糖皮质激素受体可部分逆转B细胞发育的停止,但不能逆转受损的胸腺T细胞的发育为了进一步明确下丘脑-垂体-肾上腺轴活化后释放的糖皮质激素对外周免疫细胞的发育,我们用糖皮质激素受体阻断剂阻断下丘脑-垂体-肾上腺轴并观察感染后B细胞和胸腺T细胞的发育。我们先观察了骨髓中B细胞的发育,结果发现,糖皮质激素受体阻断剂RU486处理感染组骨髓发育中B细胞的比例较溶剂处理感染组增加。进一步,我们分析了发育中B细胞的两个亚群：pro-/pre-B细胞和不成熟B细胞。结果表明,RU486处理感染组pro-/pre-B细胞占发育中B细胞的比例较溶剂处理感染组显著增加；RU486处理感染组不成熟B细胞占发育中B细胞比例较溶剂处理感染组无显著差异。进一步观察脾脏中B细胞的发育,结果显示,RU486处理感染组脾脏发育中B细胞的比例较溶剂处理感染组增加。以上结果提示广州管圆线虫感染导致下丘脑-垂体-肾上腺轴活化释放的糖皮质激素,与B细胞发育受阻从而导致B细胞输出下降有关。除了评价阻断下丘脑-垂体-肾上腺轴对B细胞发育的影响,我们同时检测RU486阻断下丘脑-垂体-肾上腺轴对胸腺T细胞发育的影响。结果发现, RU486处理感染组胸腺CD4+CD8+T比例较溶剂处理感染组无显著差异。提示RU486阻断下丘脑-垂体-肾上腺轴并不能逆转CD4+CD8+T比例的降低,胸腺T细胞发育受阻可能是下丘脑-垂体-肾上腺轴活化之外的机制所导致。综上所述,本研究证实了广州管圆线虫感染后引起的脑损伤会对免疫系统产生抑制效应,并初步阐明下丘脑-垂体-肾上腺轴活化在其中的效应机制。研究结果进一步丰富了对广州管圆线虫病致病机制的认识,也对指导广州管圆线虫病的临床治疗,提供了重要的参考依据。
[Abstract]:Guangzhou tube nematode is an important pathogen causing eosinophilic meningitis and meningoencephalitis. Human is an unsuitable host of the Guangzhou tube nematode, which infects the intermediate host of the third stage larva of the Guangzhou tube nematode. The infective larvae can pass through the intestinal wall and enter the blood circulation system and move with the blood flow in the body. A neurotropic, multiple invasion of the central nervous system. The disease is characterized by a rise in eosinophils in the cerebrospinal fluid, with the exception of the brain and meninges in the cerebellum, brain stem, spinal cord, or eyeball. The main lesions are cerebral congestion, bleeding, mechanical damage to the brain and granuloma in the brain. In recent years, the central Lodge of the Guangzhou nematode nematode The main species of snail and agate in the southern part of China are propagated in the southern part of our country, and people are addicted to living and hunting, resulting in the outbreak or epidemic of the disease in Guangzhou. The disease has been included in the new infectious disease. The inflammatory response can lead to the aggravation of the nervous system symptoms. Therefore, it is of great significance to understand the immune response mechanism of the Guangzhou tube nematode infection to the disease of Guangzhou. The study shows that the central nervous system and the immune system interact in a complex way. In the ideal case, the stress bar is ideal. Inflammatory and anti-inflammatory responses are balanced, conducive to healing and containment of the pathogen, while preventing excessive inflammation or severe immunosuppression. However, in the absence of systemic inflammation, the anti-inflammatory response triggered by local inflammatory cytokine proliferation may be harmful in the absence of systemic inflammation, as this will reduce the body's prevention. It has been found that the central nervous system injury has a profound effect on the immune function, such as stroke, traumatic brain injury or spinal cord injury, all of the patients show impaired immune function, including the decrease in the number of lymphocytes in the peripheral blood, the atrophy of the spleen and thymus, and the damage of T cell activity. It was found that the expression of major histocompatibility complex II molecules in the circulating monocytes was downregulated in the patients with acute central nervous system damage, and the ability to stimulate monocytes in vitro was greatly reduced. The clinical manifestation of this immunosuppression was the high incidence of systemic infection, especially in the lungs, after the injury of the brain. The results of these studies suggest that brain damage can lead to peripheral immunosuppression. The results of these studies suggest that brain damage can lead to peripheral immunosuppression. The larvae of Guangzhou's tube nematode can be moved to the brain after invasion to the brain, causing mechanical damage to brain tissue, occupying and releasing metabolites to damage the central nervous system. Based on brain damage and The close link of peripheral immunosuppression, we propose the following hypothesis: Guangzhou tube nematode larvae invade the brain damage to the central nervous system and cause local inflammatory cytokine diffusion to stimulate the peripheral immune suppression and increase the opportunity for secondary infection of the body. In order to verify the hypothesis, the mouse Guangzhou tube nematode In the infection model, we first observed the changes in the thymus and spleen of the immune organs of Guangzhou, and evaluated the changes in the number and function of the peripheral lymphocyte subsets. Secondly, we explored the number of peripheral lymphocytes after the two aspects of the apoptosis and the obstruction of the cell development in Guangzhou. Again, we studied the expression of inflammatory cytokine, anti-inflammatory cytokine and chemokine in the brain tissue of Guangzhou, and observed the changes in the hypothalamus pituitary adrenal (HPA) axis of the central nervous system and the peripheral immune system. Finally, we confirmed that the central nervous system of Guangzhou tube nematode was infected with the central nervous system. The main results were as follows: 1. the thymus and spleen of mice were obviously atrophied and the number of mononuclear cells decreased in 21 days after the infection induced atrophy of the immune organs and the number of lymphocytes in Guangzhou, and the number of mononuclear cells was also reduced, suggesting the infection of Guangzhou tube nematode. The immune organs of mice were affected. In order to investigate the causes of the atrophy of the thymus and spleen after infection, we detected the changes in the spleen and peripheral blood cell subsets in mice by flow cytometry. The results showed that the number of B cells, T cells, CD4+T cells, CD8+T, Thl and NK cells in the spleen and peripheral blood after infection was significantly higher than that of the control group. .2. Guangzhou tube nematode infection reduces cellular and humoral immunity in order to prove that the immune function of lymphocyte is also affected by the infection of T. We further study the proliferation of T lymphocytes and the ability to secrete cytokines and B cells to produce antibodies. The results show that the infected mouse T cells are infected after infection. The ability of proliferation was lower than that of the control group; using mitogen stimulated peripheral blood cells, the result of ELISA detection in the culture supernatant showed that the infection of Guangzhou tube nematode induced low level of IFN- gamma, TNF- alpha and high level IL-4, and the non specific antigen OVA immunized the infected mice and the control mice of the Guangzhou tube nematode, and detected the OVA-Ig. The level of Gl antibody expression. The results showed that the level of OVA-IgGl antibody in the mice after infection was lower than that of the control mice. It suggested that the infection of Guangzhou tube nematode decreased the ability to produce antibodies in mouse B cells. In order to further confirm the results, we observed the pathology of the lungs and the blood culture of mice. The results showed that the results showed that the mice were cultured. A large number of inflammatory cells were infiltrated in the lungs of the mice after infection, showing pneumonia. The same blood on the blood agar plate showed infection after infection, indicating that the infection of Guangzhou tube nematode induced the immune suppression of.3. Guangzhou tube nematode infection enhanced NK cell killing function because Guangzhou tube nematode infection reduced cell immunity The immune function of pestilence and humoral immunity increased the risk of infection in mice, so we further evaluated the effect of Guangzhou tube nematode infection on NK cell function. We stimulated 24 h by adding or without exogenous IL-12, and ELISA was used to detect the expression level of IFN- gamma and TNF- alpha in the culture supernatant. The results showed that IL-12 treatment was the result of IL-12 treatment. The infected mouse NK cells produced a higher level of IFN- gamma than the control mice. No matter whether there were exogenous IL-12 or not, the infected mice and the control mice of the Guangzhou tube could only induce the low level of TNF- beta in the NK cells. Further we evaluated the effect of infection on the cytotoxicity of NK cells. The results showed that the infected NK cells were compared with the control group. These results suggest that the infection of Guangzhou tube nematode, although the number of NK cells decreased, increased the killing function of NK cells,.4., and the decrease of B and T cells after the infection of Guangzhou tube nematode is not caused by apoptosis, but the number of B and T cells after a large number of apoptotic infection after the infection of NK cells is the number of NK cells. In order to further clarify whether the cell number decline was caused by apoptosis, we detected the proportion of 7AAD-AnnexinV+ on the surface of these cells by flow cytometry. The results showed that, compared with the control group, the 7AAD "Annexin V+ ratio" on the surface of the infected cells did not increase.Caspase 3 as the ultimate executor of apoptosis, and was a heavy detection of apoptosis. To further confirm the results, B220- cells and CD3- cells were sorted with magnetic beads. The expression of barium Cleaved Caspase 3 was detected by estern blot. The results showed that the expression of Cleaved Caspase 3 was not detected after infection, and the number of B cells and T cells decreased after the infection of Guangzhou tube nematode. It was not caused by apoptosis. Similarly, to determine whether the number of NK cells decreased by apoptosis, we detected the proportion of 7AAD "Annexin V+" on the surface of NK cells and the separation of DX5+ cells by magnetic beads, and the expression of Cleaved Caspase 3 with estern blot. The results showed that the infection was compared with the control group. The expression of 7AAD- Annexin V+ and Cleaved Caspase 3 on the surface of the post NK cells increased, suggesting that the decrease of NK number was associated with the decrease of NK number and the apoptosis of NK cell, which inhibited the occurrence of B cells in the bone marrow and the damage of T cells in the thymus gland, and the decrease of the number of B cells after the development of the thymus in the thymus gland. It is due to cell apoptosis, we speculate whether or not all or part of the developing B cell subsets are lost during the development of B cells. In order to verify this conjecture, we detected the proportion of B cells in the development of bone marrow. The results showed that the proportion of B cells in the development of bone marrow after infection was lower than that of the control group. Further, we analyzed it. Two subgroups of B cells in the development of bone marrow: pro-/pre-B cells and immature B cells. The results showed that the proportion of pro-/pre-B cells in the bone marrow after infection was significantly lower than that of the control group, while the proportion of immature B cells in the bone marrow after infection accounted for the increase of the proportion of B cells in the developmental B cells, suggesting that the infection of Guangzhou tube nematode was caused by infection. B cells decline in the development of bone marrow, which is mainly affected by pro-/pre-B cells. We know that after the rearrangement of the Ig H chain and the L chain gene, the developing B cells in the bone marrow enter the immature B cell stage and then migrate to the peripheral B cells in the transitional period. We have detected the B cells and maturation of the spleen in the development of the spleen. The proportion of B cells showed that the mature B cells decreased at 21 days after infection. At the same time, the proportion of B cells in the development of spleen was significantly lower than that of the control group. Further, we analyzed the 3 subgroups of the developing B cells: TR1, TR2 and TR3 subgroups. The results showed that the proportion of TR1 in the developing B cells gradually reduced.TR2 with the increase of the infection time. The proportion of TR3 to the developing B cells increased with the increase of the time of infection. It suggests that the infection of Guangzhou tube nematode leads to the decrease of B cells in the development of the spleen, which is mainly affected by the TR1 cell subgroup. The above results suggest that the output of B cells is affected by the infection of the Guangzhou tube nematode, which leads to the decline of the output of B cells. Guangzhou tube The decrease in the number of T cells after the infection of the nematode is also not caused by apoptosis, and there is a serious atrophy in the thymus after infection. We speculate whether the T cells are blocked in the development of the thymus. Therefore, we detected the thymus T cell subgroup by flow cytometry, and the result showed that the proportion of CD4+CD8+T cells after infection was lower than that of the control group. On the contrary, the proportion of CD4-CD8-T cells, CD4+CD8-T cells and CD4-CD8+T cells increased significantly with the increase of infection time. Further we observed the number of T cell subgroups in the thymus. The results showed that the number of CD4CD8T cells and CD4 "CD8+T cells" after infection was not different from that of the control group; but after infection, CD4+CD was found to be CD4+CD. The number of 8+T cells was significantly lower than that in the control group. In order to further clarify whether the number of cells decreased by apoptosis, we detected the expression of 7AAD "Annexin V+" on the surface of CD4+CD8+T cells by flow cytometry. The results showed that the proportion of 7AAD-Annexin V+ on the surface of CD4+CD8+T cells after infection was gradually increased compared with the control group. The results of the above results suggest that the ratio of 7AAD-Annexin V+ to the surface of CD4+CD8+T cells after infection is gradually increased. The decrease of the number of thymus cells and the number of peripheral T cells and the decrease of the number of peripheral T cells and the decrease of CD4+CD8+T cell apoptosis after the infection of the Caenorhabditis elegans in Guangzhou, the decrease of the number of lymphocytes and the activation of the hypothalamus pituitary adrenal axis in the brain, and the invasion of the nervous system by the activation of the larva of the hypothalamus pituitary adrenal axis of the Guangzhou tube nematode. In order to determine whether the decrease of the number of lymphocytes caused by the infection of the nematode in Guangzhou was caused by brain damage, we used albendazole to observe the influence of the peripheral lymphocyte subsets after infection. The results showed that the size of the thymus and spleen in the treatment group was more than that of the normal control group 21 days after the infection. There was no significant difference. The proportion of B lymphocyte, T cell, CD4+T cell, CD8+T cell and NK cell of spleen lymphocyte subgroup after infection were higher.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R532.1;R741
，
本文编号：2119022
[Abstract]:Guangzhou tube nematode is an important pathogen causing eosinophilic meningitis and meningoencephalitis. Human is an unsuitable host of the Guangzhou tube nematode, which infects the intermediate host of the third stage larva of the Guangzhou tube nematode. The infective larvae can pass through the intestinal wall and enter the blood circulation system and move with the blood flow in the body. A neurotropic, multiple invasion of the central nervous system. The disease is characterized by a rise in eosinophils in the cerebrospinal fluid, with the exception of the brain and meninges in the cerebellum, brain stem, spinal cord, or eyeball. The main lesions are cerebral congestion, bleeding, mechanical damage to the brain and granuloma in the brain. In recent years, the central Lodge of the Guangzhou nematode nematode The main species of snail and agate in the southern part of China are propagated in the southern part of our country, and people are addicted to living and hunting, resulting in the outbreak or epidemic of the disease in Guangzhou. The disease has been included in the new infectious disease. The inflammatory response can lead to the aggravation of the nervous system symptoms. Therefore, it is of great significance to understand the immune response mechanism of the Guangzhou tube nematode infection to the disease of Guangzhou. The study shows that the central nervous system and the immune system interact in a complex way. In the ideal case, the stress bar is ideal. Inflammatory and anti-inflammatory responses are balanced, conducive to healing and containment of the pathogen, while preventing excessive inflammation or severe immunosuppression. However, in the absence of systemic inflammation, the anti-inflammatory response triggered by local inflammatory cytokine proliferation may be harmful in the absence of systemic inflammation, as this will reduce the body's prevention. It has been found that the central nervous system injury has a profound effect on the immune function, such as stroke, traumatic brain injury or spinal cord injury, all of the patients show impaired immune function, including the decrease in the number of lymphocytes in the peripheral blood, the atrophy of the spleen and thymus, and the damage of T cell activity. It was found that the expression of major histocompatibility complex II molecules in the circulating monocytes was downregulated in the patients with acute central nervous system damage, and the ability to stimulate monocytes in vitro was greatly reduced. The clinical manifestation of this immunosuppression was the high incidence of systemic infection, especially in the lungs, after the injury of the brain. The results of these studies suggest that brain damage can lead to peripheral immunosuppression. The results of these studies suggest that brain damage can lead to peripheral immunosuppression. The larvae of Guangzhou's tube nematode can be moved to the brain after invasion to the brain, causing mechanical damage to brain tissue, occupying and releasing metabolites to damage the central nervous system. Based on brain damage and The close link of peripheral immunosuppression, we propose the following hypothesis: Guangzhou tube nematode larvae invade the brain damage to the central nervous system and cause local inflammatory cytokine diffusion to stimulate the peripheral immune suppression and increase the opportunity for secondary infection of the body. In order to verify the hypothesis, the mouse Guangzhou tube nematode In the infection model, we first observed the changes in the thymus and spleen of the immune organs of Guangzhou, and evaluated the changes in the number and function of the peripheral lymphocyte subsets. Secondly, we explored the number of peripheral lymphocytes after the two aspects of the apoptosis and the obstruction of the cell development in Guangzhou. Again, we studied the expression of inflammatory cytokine, anti-inflammatory cytokine and chemokine in the brain tissue of Guangzhou, and observed the changes in the hypothalamus pituitary adrenal (HPA) axis of the central nervous system and the peripheral immune system. Finally, we confirmed that the central nervous system of Guangzhou tube nematode was infected with the central nervous system. The main results were as follows: 1. the thymus and spleen of mice were obviously atrophied and the number of mononuclear cells decreased in 21 days after the infection induced atrophy of the immune organs and the number of lymphocytes in Guangzhou, and the number of mononuclear cells was also reduced, suggesting the infection of Guangzhou tube nematode. The immune organs of mice were affected. In order to investigate the causes of the atrophy of the thymus and spleen after infection, we detected the changes in the spleen and peripheral blood cell subsets in mice by flow cytometry. The results showed that the number of B cells, T cells, CD4+T cells, CD8+T, Thl and NK cells in the spleen and peripheral blood after infection was significantly higher than that of the control group. .2. Guangzhou tube nematode infection reduces cellular and humoral immunity in order to prove that the immune function of lymphocyte is also affected by the infection of T. We further study the proliferation of T lymphocytes and the ability to secrete cytokines and B cells to produce antibodies. The results show that the infected mouse T cells are infected after infection. The ability of proliferation was lower than that of the control group; using mitogen stimulated peripheral blood cells, the result of ELISA detection in the culture supernatant showed that the infection of Guangzhou tube nematode induced low level of IFN- gamma, TNF- alpha and high level IL-4, and the non specific antigen OVA immunized the infected mice and the control mice of the Guangzhou tube nematode, and detected the OVA-Ig. The level of Gl antibody expression. The results showed that the level of OVA-IgGl antibody in the mice after infection was lower than that of the control mice. It suggested that the infection of Guangzhou tube nematode decreased the ability to produce antibodies in mouse B cells. In order to further confirm the results, we observed the pathology of the lungs and the blood culture of mice. The results showed that the results showed that the mice were cultured. A large number of inflammatory cells were infiltrated in the lungs of the mice after infection, showing pneumonia. The same blood on the blood agar plate showed infection after infection, indicating that the infection of Guangzhou tube nematode induced the immune suppression of.3. Guangzhou tube nematode infection enhanced NK cell killing function because Guangzhou tube nematode infection reduced cell immunity The immune function of pestilence and humoral immunity increased the risk of infection in mice, so we further evaluated the effect of Guangzhou tube nematode infection on NK cell function. We stimulated 24 h by adding or without exogenous IL-12, and ELISA was used to detect the expression level of IFN- gamma and TNF- alpha in the culture supernatant. The results showed that IL-12 treatment was the result of IL-12 treatment. The infected mouse NK cells produced a higher level of IFN- gamma than the control mice. No matter whether there were exogenous IL-12 or not, the infected mice and the control mice of the Guangzhou tube could only induce the low level of TNF- beta in the NK cells. Further we evaluated the effect of infection on the cytotoxicity of NK cells. The results showed that the infected NK cells were compared with the control group. These results suggest that the infection of Guangzhou tube nematode, although the number of NK cells decreased, increased the killing function of NK cells,.4., and the decrease of B and T cells after the infection of Guangzhou tube nematode is not caused by apoptosis, but the number of B and T cells after a large number of apoptotic infection after the infection of NK cells is the number of NK cells. In order to further clarify whether the cell number decline was caused by apoptosis, we detected the proportion of 7AAD-AnnexinV+ on the surface of these cells by flow cytometry. The results showed that, compared with the control group, the 7AAD "Annexin V+ ratio" on the surface of the infected cells did not increase.Caspase 3 as the ultimate executor of apoptosis, and was a heavy detection of apoptosis. To further confirm the results, B220- cells and CD3- cells were sorted with magnetic beads. The expression of barium Cleaved Caspase 3 was detected by estern blot. The results showed that the expression of Cleaved Caspase 3 was not detected after infection, and the number of B cells and T cells decreased after the infection of Guangzhou tube nematode. It was not caused by apoptosis. Similarly, to determine whether the number of NK cells decreased by apoptosis, we detected the proportion of 7AAD "Annexin V+" on the surface of NK cells and the separation of DX5+ cells by magnetic beads, and the expression of Cleaved Caspase 3 with estern blot. The results showed that the infection was compared with the control group. The expression of 7AAD- Annexin V+ and Cleaved Caspase 3 on the surface of the post NK cells increased, suggesting that the decrease of NK number was associated with the decrease of NK number and the apoptosis of NK cell, which inhibited the occurrence of B cells in the bone marrow and the damage of T cells in the thymus gland, and the decrease of the number of B cells after the development of the thymus in the thymus gland. It is due to cell apoptosis, we speculate whether or not all or part of the developing B cell subsets are lost during the development of B cells. In order to verify this conjecture, we detected the proportion of B cells in the development of bone marrow. The results showed that the proportion of B cells in the development of bone marrow after infection was lower than that of the control group. Further, we analyzed it. Two subgroups of B cells in the development of bone marrow: pro-/pre-B cells and immature B cells. The results showed that the proportion of pro-/pre-B cells in the bone marrow after infection was significantly lower than that of the control group, while the proportion of immature B cells in the bone marrow after infection accounted for the increase of the proportion of B cells in the developmental B cells, suggesting that the infection of Guangzhou tube nematode was caused by infection. B cells decline in the development of bone marrow, which is mainly affected by pro-/pre-B cells. We know that after the rearrangement of the Ig H chain and the L chain gene, the developing B cells in the bone marrow enter the immature B cell stage and then migrate to the peripheral B cells in the transitional period. We have detected the B cells and maturation of the spleen in the development of the spleen. The proportion of B cells showed that the mature B cells decreased at 21 days after infection. At the same time, the proportion of B cells in the development of spleen was significantly lower than that of the control group. Further, we analyzed the 3 subgroups of the developing B cells: TR1, TR2 and TR3 subgroups. The results showed that the proportion of TR1 in the developing B cells gradually reduced.TR2 with the increase of the infection time. The proportion of TR3 to the developing B cells increased with the increase of the time of infection. It suggests that the infection of Guangzhou tube nematode leads to the decrease of B cells in the development of the spleen, which is mainly affected by the TR1 cell subgroup. The above results suggest that the output of B cells is affected by the infection of the Guangzhou tube nematode, which leads to the decline of the output of B cells. Guangzhou tube The decrease in the number of T cells after the infection of the nematode is also not caused by apoptosis, and there is a serious atrophy in the thymus after infection. We speculate whether the T cells are blocked in the development of the thymus. Therefore, we detected the thymus T cell subgroup by flow cytometry, and the result showed that the proportion of CD4+CD8+T cells after infection was lower than that of the control group. On the contrary, the proportion of CD4-CD8-T cells, CD4+CD8-T cells and CD4-CD8+T cells increased significantly with the increase of infection time. Further we observed the number of T cell subgroups in the thymus. The results showed that the number of CD4CD8T cells and CD4 "CD8+T cells" after infection was not different from that of the control group; but after infection, CD4+CD was found to be CD4+CD. The number of 8+T cells was significantly lower than that in the control group. In order to further clarify whether the number of cells decreased by apoptosis, we detected the expression of 7AAD "Annexin V+" on the surface of CD4+CD8+T cells by flow cytometry. The results showed that the proportion of 7AAD-Annexin V+ on the surface of CD4+CD8+T cells after infection was gradually increased compared with the control group. The results of the above results suggest that the ratio of 7AAD-Annexin V+ to the surface of CD4+CD8+T cells after infection is gradually increased. The decrease of the number of thymus cells and the number of peripheral T cells and the decrease of the number of peripheral T cells and the decrease of CD4+CD8+T cell apoptosis after the infection of the Caenorhabditis elegans in Guangzhou, the decrease of the number of lymphocytes and the activation of the hypothalamus pituitary adrenal axis in the brain, and the invasion of the nervous system by the activation of the larva of the hypothalamus pituitary adrenal axis of the Guangzhou tube nematode. In order to determine whether the decrease of the number of lymphocytes caused by the infection of the nematode in Guangzhou was caused by brain damage, we used albendazole to observe the influence of the peripheral lymphocyte subsets after infection. The results showed that the size of the thymus and spleen in the treatment group was more than that of the normal control group 21 days after the infection. There was no significant difference. The proportion of B lymphocyte, T cell, CD4+T cell, CD8+T cell and NK cell of spleen lymphocyte subgroup after infection were higher.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R532.1;R741
，
本文编号：2119022
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