本文選題：日本血吸蟲 + 肝臟病理　； 參考：《南京醫(yī)科大學(xué)》2013年博士論文

【摘要】：血吸蟲病是當(dāng)前我國乃至世界最嚴(yán)重的寄生蟲病之一,全球感染人數(shù)超過2億。在我國,日本血吸蟲病是一種流行廣泛,危害嚴(yán)重的傳染病,迄今仍然是我國主要公共衛(wèi)生問題之一。日本血吸蟲感染后,沉積在肝臟內(nèi)的蟲卵引起以蟲卵肉芽腫為主的免疫病理損害,進(jìn)一步引起進(jìn)行性發(fā)展的肝臟纖維化,并最終導(dǎo)致肝硬化、門脈高壓癥等,是對宿主最嚴(yán)重的免疫損傷,甚至危及生命。因此,監(jiān)測血吸蟲病人肝臟免疫病理損傷進(jìn)程、研究血吸蟲病肝臟免疫病理損傷的機(jī)制對于治療血吸蟲病人是非常迫切和必要的。然而,現(xiàn)行的以B超檢測為主的方法因其不能檢測出血吸蟲引起的早期肝臟纖維化、而且使用受偏遠(yuǎn)的農(nóng)村和山區(qū)地區(qū)經(jīng)濟(jì)和電力供應(yīng)等方面的因素限制以及不適用于大規(guī)模的流行病學(xué)篩查等,導(dǎo)致其在現(xiàn)場使用具有很大的局限性,因此研發(fā)一種較為簡單的免疫學(xué)方法,在血吸蟲感染早期即可檢測宿主對血吸蟲感染免疫應(yīng)答的強(qiáng)弱、叢而在血吸蟲感染早期即可估測特定患者今后可能引起免疫病理損害的進(jìn)展速度和/或程度,或用于感染后動態(tài)監(jiān)測患者免疫病理損害進(jìn)程與程度,并可能在流行區(qū)人群中低成本、大規(guī)模地反復(fù)應(yīng)用的檢測方法是非常重要的。 我們在實(shí)驗中發(fā)現(xiàn),日本血吸蟲感染小鼠的血清SjHSP60-IgG及其亞型IgG1抗體滴度與對應(yīng)的肝臟單個蟲卵肉芽腫面積呈正相關(guān)；日本血吸蟲感染家兔的血清SjHSP60-IgG抗體滴度與對應(yīng)的肝臟單個蟲卵肉芽腫面積也呈正相關(guān),且在家兔的慢性感染模型中(感染23周)也觀察到家兔肝臟中的膠原纖維Ⅲ和a-SMA的表達(dá)水平與血清中的SjHSP60-IgG抗體滴度呈正相關(guān)。在注射四氯化碳模擬肝臟綜合免疫病理損傷的日本血吸蟲感染小鼠中發(fā)現(xiàn),其血清SjHSP60-IgG抗體水平明顯高于單純?nèi)毡狙x感染小鼠。在血吸病流行區(qū)糞檢陽性的日本血吸蟲病人中發(fā)現(xiàn)血清SjHSP60-IgG及其亞型IgG1抗體滴度與各對應(yīng)患者的肝臟超聲病理指標(biāo)亦呈正相關(guān)。上述結(jié)果均表明日本血吸蟲感染宿主的血清SjHSP60-IgG及其亞型IgG1抗體水平與肝臟肉芽腫和纖維化程度呈正相關(guān),從而提示血清SjHSP60-IgG及其亞型IgG1抗體水平可以作為評價日本血吸蟲患者肝臟病理程度的一個指標(biāo),具有潛在的臨床應(yīng)用價值。 然而,宿主血清中SjHSP60抗體水平與其肝臟免疫病理損傷程度呈正相關(guān)的免疫學(xué)機(jī)制還不清楚。在我們的后續(xù)研究中發(fā)現(xiàn),日本血吸蟲感染的Tfh細(xì)胞缺陷小鼠(ICOSL敲除鼠)的血清SjHSP60抗體水平極低,從而提示SjHSP60抗體產(chǎn)生依賴于Tfh細(xì)胞。為闡明是否是日本血吸蟲感染過程中誘導(dǎo)產(chǎn)生的Tfh細(xì)胞在促進(jìn)SjHSP60抗體生成的同時也促進(jìn)肝臟病理的形成,從而導(dǎo)致了血清SjHSP60抗體水平與肝臟免疫病理損傷程度呈正相關(guān),我們進(jìn)行了深入的機(jī)制研究,并獲得了如下結(jié)果：我們發(fā)現(xiàn)日本日本血吸蟲感染小鼠8周后,隨著肝臟蟲卵肉芽腫的發(fā)展,小鼠脾臟、淋巴結(jié)和肝臟中Tfh細(xì)胞比例顯著升高,初步提示了Tfh細(xì)胞可能參與了肝臟蟲卵肉芽腫的形成。隨后我們發(fā)現(xiàn),Tfh細(xì)胞缺陷小鼠在感染日本血吸蟲后肝臟蟲卵肉芽腫反應(yīng)顯著減輕,而其接受過繼轉(zhuǎn)移的Tfh細(xì)胞后,蟲卵肉芽腫反應(yīng)加重并回復(fù)至正常水平,從而證明Tfh細(xì)胞確能促進(jìn)蟲卵肉芽腫的形成。我們還發(fā)現(xiàn)Tfh細(xì)胞可以被直接募集到肝臟中參與促進(jìn)蟲卵肉芽腫的形成。 隨后,我們探討了日本血吸蟲感染小鼠體內(nèi)Tfh細(xì)胞形成的相關(guān)機(jī)制,首次發(fā)現(xiàn)巨噬細(xì)胞可以通過細(xì)胞-細(xì)胞接觸的方式誘導(dǎo)CD4+T細(xì)胞分化成Tfh細(xì)胞,并且巨噬細(xì)胞表面的ICOSL分子對于誘導(dǎo)Tfh細(xì)胞是必須的。我們還發(fā)現(xiàn)CD40/CD40L信號可以通過上調(diào)巨噬細(xì)胞表面的ICOSL的表達(dá)而促進(jìn)Tfh的誘導(dǎo)。 總之,本研究首次發(fā)現(xiàn)了日本血吸蟲感染小鼠的巨噬細(xì)胞通過細(xì)胞-細(xì)胞接觸、并依賴于其表面ICOSL分子誘導(dǎo)Tfh細(xì)胞產(chǎn)生,來參與蟲卵肉芽腫形成并促進(jìn)血清SjHSP60抗體產(chǎn)生,最終導(dǎo)致了宿主血清SjHSP60抗體水平與肝臟免疫病理損傷程度呈正相關(guān)。我們的研究結(jié)果不僅解釋了SjHSP60抗體和肝臟免疫病理呈相關(guān)性的免疫學(xué)現(xiàn)象,還首次發(fā)現(xiàn)了Tfh細(xì)胞參與肉芽腫形成這一新功能并闡述了巨噬細(xì)胞誘導(dǎo)Tfh細(xì)胞形成的新機(jī)制,從而有助于更好地了解和認(rèn)識Tfh細(xì)胞在生理和疾病中的作用,也為以Tfh細(xì)胞誘導(dǎo)過程與功能發(fā)揮為干預(yù)靶標(biāo)的新型治療方法的研發(fā)奠定理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R532.21

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