日本血吸虫病患者肝脏病理损害的免疫学诊断方法的建立及相关机制探讨

发布时间：2018-05-24 04:14

本文选题：日本血吸虫 + 肝脏病理　；参考：《南京医科大学》2013年博士论文

【摘要】：血吸虫病是当前我国乃至世界最严重的寄生虫病之一,全球感染人数超过2亿。在我国,日本血吸虫病是一种流行广泛,危害严重的传染病,迄今仍然是我国主要公共卫生问题之一。日本血吸虫感染后,沉积在肝脏内的虫卵引起以虫卵肉芽肿为主的免疫病理损害,进一步引起进行性发展的肝脏纤维化,并最终导致肝硬化、门脉高压症等,是对宿主最严重的免疫损伤,甚至危及生命。因此,监测血吸虫病人肝脏免疫病理损伤进程、研究血吸虫病肝脏免疫病理损伤的机制对于治疗血吸虫病人是非常迫切和必要的。然而,现行的以B超检测为主的方法因其不能检测出血吸虫引起的早期肝脏纤维化、而且使用受偏远的农村和山区地区经济和电力供应等方面的因素限制以及不适用于大规模的流行病学筛查等,导致其在现场使用具有很大的局限性,因此研发一种较为简单的免疫学方法,在血吸虫感染早期即可检测宿主对血吸虫感染免疫应答的强弱、丛而在血吸虫感染早期即可估测特定患者今后可能引起免疫病理损害的进展速度和/或程度,或用于感染后动态监测患者免疫病理损害进程与程度,并可能在流行区人群中低成本、大规模地反复应用的检测方法是非常重要的。我们在实验中发现,日本血吸虫感染小鼠的血清SjHSP60-IgG及其亚型IgG1抗体滴度与对应的肝脏单个虫卵肉芽肿面积呈正相关；日本血吸虫感染家兔的血清SjHSP60-IgG抗体滴度与对应的肝脏单个虫卵肉芽肿面积也呈正相关,且在家兔的慢性感染模型中(感染23周)也观察到家兔肝脏中的胶原纤维Ⅲ和a-SMA的表达水平与血清中的SjHSP60-IgG抗体滴度呈正相关。在注射四氯化碳模拟肝脏综合免疫病理损伤的日本血吸虫感染小鼠中发现,其血清SjHSP60-IgG抗体水平明显高于单纯日本血吸虫感染小鼠。在血吸病流行区粪检阳性的日本血吸虫病人中发现血清SjHSP60-IgG及其亚型IgG1抗体滴度与各对应患者的肝脏超声病理指标亦呈正相关。上述结果均表明日本血吸虫感染宿主的血清SjHSP60-IgG及其亚型IgG1抗体水平与肝脏肉芽肿和纤维化程度呈正相关,从而提示血清SjHSP60-IgG及其亚型IgG1抗体水平可以作为评价日本血吸虫患者肝脏病理程度的一个指标,具有潜在的临床应用价值。然而,宿主血清中SjHSP60抗体水平与其肝脏免疫病理损伤程度呈正相关的免疫学机制还不清楚。在我们的后续研究中发现,日本血吸虫感染的Tfh细胞缺陷小鼠(ICOSL敲除鼠)的血清SjHSP60抗体水平极低,从而提示SjHSP60抗体产生依赖于Tfh细胞。为阐明是否是日本血吸虫感染过程中诱导产生的Tfh细胞在促进SjHSP60抗体生成的同时也促进肝脏病理的形成,从而导致了血清SjHSP60抗体水平与肝脏免疫病理损伤程度呈正相关,我们进行了深入的机制研究,并获得了如下结果：我们发现日本日本血吸虫感染小鼠8周后,随着肝脏虫卵肉芽肿的发展,小鼠脾脏、淋巴结和肝脏中Tfh细胞比例显著升高,初步提示了Tfh细胞可能参与了肝脏虫卵肉芽肿的形成。随后我们发现,Tfh细胞缺陷小鼠在感染日本血吸虫后肝脏虫卵肉芽肿反应显著减轻,而其接受过继转移的Tfh细胞后,虫卵肉芽肿反应加重并回复至正常水平,从而证明Tfh细胞确能促进虫卵肉芽肿的形成。我们还发现Tfh细胞可以被直接募集到肝脏中参与促进虫卵肉芽肿的形成。随后,我们探讨了日本血吸虫感染小鼠体内Tfh细胞形成的相关机制,首次发现巨噬细胞可以通过细胞-细胞接触的方式诱导CD4+T细胞分化成Tfh细胞,并且巨噬细胞表面的ICOSL分子对于诱导Tfh细胞是必须的。我们还发现CD40/CD40L信号可以通过上调巨噬细胞表面的ICOSL的表达而促进Tfh的诱导。总之,本研究首次发现了日本血吸虫感染小鼠的巨噬细胞通过细胞-细胞接触、并依赖于其表面ICOSL分子诱导Tfh细胞产生,来参与虫卵肉芽肿形成并促进血清SjHSP60抗体产生,最终导致了宿主血清SjHSP60抗体水平与肝脏免疫病理损伤程度呈正相关。我们的研究结果不仅解释了SjHSP60抗体和肝脏免疫病理呈相关性的免疫学现象,还首次发现了Tfh细胞参与肉芽肿形成这一新功能并阐述了巨噬细胞诱导Tfh细胞形成的新机制,从而有助于更好地了解和认识Tfh细胞在生理和疾病中的作用,也为以Tfh细胞诱导过程与功能发挥为干预靶标的新型治疗方法的研发奠定理论基础。
[Abstract]:Schistosomiasis is one of the most serious parasitic diseases in our country and in the world. The number of infections in the world is more than 200 million. In China, schistosomiasis is a widespread and serious infectious disease. So far, it is still one of the major public health problems in China. After Schistosoma japonicum infection, the eggs deposited in the liver are caused by eggs granulation. The main immune pathological damage, further causing progressive development of liver fibrosis, and eventually leading to liver cirrhosis, portal hypertension and so on, is the most serious immune injury to the host, even endangers life. Therefore, monitoring the liver immune pathological damage process in Schistosoma patients and studying the mechanism of liver immuno pathological injury of schistosomiasis for treatment of the treatment of schistosomiasis. Patients with Schistosoma are very urgent and necessary. However, the current B-mode detection method is due to its failure to detect early liver fibrosis caused by haemorrhage and the use of factors such as economic and electricity supply from remote rural and mountainous areas, as well as large-scale epidemiological screening, and so on. Its use in the field is very limited, so a relatively simple immunological method is developed to detect the immune response of the host to Schistosoma infection in the early stage of Schistosoma infection. In the early stages of infection, the speed and / or degree of the progress and / or the extent of the pathological damage of a specific patient may be estimated in the early stage of the infection of a particular patient. It is very important to dynamically monitor the process and degree of immune pathological damage in the patients after infection, and may be low cost in the population of the epidemic area.
In the experiment, we found that the titer of serum SjHSP60-IgG and its subtype IgG1 antibody in the mice infected with Schistosoma japonicum was positively correlated with the corresponding area of single egg granuloma of the liver, and the titer of serum SjHSP60-IgG antibody of the rabbit infected with Schistosoma japonicum was also positively correlated with the area of the corresponding liver single egg granuloma. The expression level of collagen fiber III and a-SMA in the liver of the rabbit was also positively correlated with the titer of SjHSP60-IgG antibody in the liver of the rabbit (23 weeks of infection). The serum level of SjHSP60-IgG antibody in the sera was significantly higher than that in the mice infected with Schistosoma japonicum infected by the injection of carbon tetrachloride to simulate the comprehensive liver immuno pathological injury. In mice infected with Schistosoma japonicum, it was found that the titer of serum SjHSP60-IgG and its subtype IgG1 antibody in the patients with positive Schistosoma japonicum in the epidemic area of blood sucking disease was also positively correlated with the liver ultrasonic pathological indexes of the corresponding patients. All of these results showed that the level of SjHSP60-IgG and its subtype IgG1 antibody in the blood of the host infected by Schistosoma japonicum The liver granuloma is positively correlated with the degree of fibrosis, suggesting that the level of serum SjHSP60-IgG and its subtype IgG1 antibody can be used as an index to evaluate the liver pathology of the patients with Schistosoma japonicum, which has potential clinical value.
However, the immunological mechanism that has a positive correlation between the level of SjHSP60 antibody in the host serum and the degree of liver immuno pathological damage is not clear. In our follow-up study, we found that the serum SjHSP60 antibody level of the Tfh cell deficient mice (ICOSL knockout mice) infected by Schistosoma japonicum is very low, suggesting that the SjHSP60 antibody production depends on the Tfh cells. To clarify whether the Tfh cells induced by Schistosoma japonicum infection in the process of promoting the formation of SjHSP60 antibodies also promote the formation of liver pathology, resulting in a positive correlation between the level of serum SjHSP60 antibody and the degree of liver immune pathological damage. We have conducted in-depth mechanism research and obtained the following results: We After 8 weeks of mice infected with Schistosoma japonicum, the proportion of Tfh cells in the spleen, lymph node and liver of mice increased significantly with the development of liver egg granuloma. It was suggested that Tfh cells may be involved in the formation of liver egg granuloma. Then, we found that the Tfh cell defect mice were infected with liver eggs after infection with Schistosoma japonicum. The response of the bud swelling was significantly reduced, and after the adoptive transfer of Tfh cells, the egg granuloma response increased and returned to the normal level, which proved that Tfh cells could promote the formation of egg granuloma. We also found that Tfh cells could be directly recruited into the liver to promote the formation of egg granuloma.
Then, we explored the mechanism of Tfh cell formation in mice infected with Schistosoma japonicum. It is the first time that macrophages can induce CD4+T cells to differentiate into Tfh cells through cell cell contact, and the ICOSL molecules on the surface of the macrophages are necessary to induce Tfh cells. We also found that CD40/CD40L signals can be used. Up regulate the expression of ICOSL on macrophage surface and promote the induction of Tfh.
In conclusion, it is the first time that the macrophages of mice infected with Schistosoma japonicum have been found through cell cell contact and dependent on their surface ICOSL molecules to induce the production of Tfh cells to participate in the formation of egg granuloma and to promote the production of serum SjHSP60 antibodies, which eventually leads to the level of serum SjHSP60 antibody and the degree of liver immuno pathological damage in the host. Our results not only explain the immunological phenomena associated with SjHSP60 antibody and liver immune pathology, but also discover the new function of Tfh cells in granuloma formation for the first time and explain the new mechanism of macrophage induced Tfh cell formation, which will help to better understand and recognize the physiology and disease of Tfh cells. The role of the disease also lays a theoretical foundation for the research and development of new therapeutic methods that target the process and function of Tfh cell induction.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R532.21

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