發(fā)布時間：2018-08-14 13:18

【摘要】：背景：血吸蟲病是由聯(lián)合國開發(fā)計劃署、世界銀行和世界衛(wèi)生組織熱帶病特別規(guī)劃署共同倡議防治的一種主要的熱帶疾病。在非洲、南美洲以及亞洲等地區(qū)流行,血吸蟲病是一項嚴重的公共衛(wèi)生問題。全球約有2億人感染血吸蟲,1.2億人有典型癥狀。血吸蟲感染早期,宿主呈現(xiàn)Th1優(yōu)勢應(yīng)答,隨后出現(xiàn)Th2免疫偏移,其負向免疫調(diào)控的機制至今尚未明了。本課題通過表達、純化蟲源性糖蛋白果糖二磷酸醛縮酶(SjFBPA),感染小鼠和刺激細胞,在體外研究SjFBPA誘導Th2免疫應(yīng)答的機制,對揭示蟲體免疫逃避、宿主免疫耐受、免疫偏移等的分子機制的研究有重要意義。目的：分析日本血吸蟲感染以及蟲源性糖蛋白果糖二磷酸醛縮酶(SjFBPA)刺激后小鼠脾臟細胞中IL-4、CD8α因子的表達量的動態(tài)變化,并探討SjFBPA與Th2免疫應(yīng)答的關(guān)系。方法：本次實驗首先通過轉(zhuǎn)化將連接產(chǎn)物轉(zhuǎn)入感受態(tài)細胞,然后小量表達菌株,電泳檢測單克隆成功表達。再通過大量搖菌表達蛋白后進行純化,得到大量高純度的FBPA蛋白。構(gòu)建血吸蟲感染小鼠模型,于模型建立后設(shè)立FBPA蛋白刺激對照組。通過FCM檢測IL-4、CD8α因子表達水平以及動物模型病理標本的改變。對FBPA蛋白誘導Th2免疫偏移的機制進行全面分析。結(jié)果：(1)通過轉(zhuǎn)化后成功培養(yǎng)出單克隆表達菌落。(2)大量表達、純化后可得到濃度達0.5mg/ml的FBPA蛋白。(3)血吸蟲感染8W后小鼠脾臟細胞懸液的IL-4、CD8α因子表達量與正常對照組小鼠脾臟細胞懸液的IL-4、CD8α因子表達量比較發(fā)生明顯變化,FBPA蛋白刺激后的小鼠脾臟細胞懸液的IL-4、CD8α因子表達量與正常對照組小鼠脾臟細胞懸液的IL-4、CD8α因子表達量比較也發(fā)生了明顯變化,差異具有統(tǒng)計學意義(P0.05)。結(jié)論：本實驗研究結(jié)果顯示蟲源性果糖二磷酸醛縮酶(SjFBPA)屬于包涵體表達蛋白,通過轉(zhuǎn)化方式可以得到單克隆表達菌,純化可得到高濃度(0.5mg/ml)蛋白。SjFBPA喇激小鼠脾臟細胞懸液后細胞因子的變化與血吸蟲感染后的小鼠脾臟細胞懸液變化趨勢相同。DC細胞分泌因子CD8α的變化趨勢也相同�？傻贸鯯jFBPA在血吸蟲感染中具有誘導Th2免疫偏移的作用,DC細胞在此過程中參與了重要的免疫反應(yīng)。
[Abstract]:Background: schistosomiasis is a major tropical disease under the joint initiative of the United Nations Development Programme (UNDP), the World Bank and the World Health Organization (WHO) Special Programme on Tropical Diseases. Schistosomiasis is a serious public health problem in Africa, South America and Asia. About 200 million people worldwide are infected with schistosomiasis and 120 million have typical symptoms. In the early stage of Schistosoma japonicum infection, the host presented a dominant response to Th1, followed by a Th2 immune shift. The mechanism of negative immune regulation has not been clarified. In this study, the expression and purification of arbuscular glycoprotein fructose diphosphate aldolase (SjFBPA),) were used to infect mice and stimulate cells. The mechanism of Th2 immune response induced by SjFBPA was studied in vitro. It is important to study the molecular mechanism of immune shift. Aim: to analyze the dynamic changes of IL-4 / CD8 偽 expression in spleen cells of mice after infection of Schistosoma japonicum and (SjFBPA) stimulation of insect-derived glycoprotein fructose diphosphate aldolase (SjFBPA), and to explore the relationship between SjFBPA and Th2 immune response. Methods: in this experiment, the ligation product was first transformed into the receptive cells, then expressed in a small number of strains, and the monoclonal expression was detected by electrophoresis. A large number of high purity FBPA proteins were obtained after purification by a large number of expressed proteins. The model of Schistosoma japonicum infected mice was established, and the control group was stimulated by FBPA protein after the establishment of the model. The expression level of IL-4 and CD8 偽 and the pathological changes of animal model were detected by FCM. The mechanism of Th2 immune migration induced by FBPA protein was analyzed. Results: (1) Monoclonal expression colonies were successfully cultured after transformation. After purification, FBPA protein with concentration up to 0.5mg/ml was obtained. (3) the expression of IL-4 and CD8 偽 in spleen cell suspension of mice infected with Schistosoma japonicum was significantly different from that in normal control mice. (3) the expression of IL-4 and CD8 偽 in spleen cell suspension of mice infected with Schistosoma japonicum was significantly changed after infection with Schistosoma japonicum for 8 weeks. The expression of IL-4 and CD8 偽 in splenic cell suspension of stimulated mice was significantly different from that in normal control group. The difference was statistically significant (P0.05). Conclusion: the results of this study showed that the insect-derived fructose diphosphate aldolase (SjFBPA) belonged to the inclusion body expression protein, and monoclonal expression bacteria could be obtained by transformation. The changes of cytokines in spleen cell suspension of SjFBPA-stimulated mice were similar to those of mice infected with Schistosoma japonicum. The change trend of CD8 偽 secreted by DC cells was the same as that in mice infected with Schistosoma japonicum. It can be concluded that SjFBPA can induce Th2 immune deviation in schistosomiasis infection. DC cells participate in the important immune response in the process of Schistosoma japonicum infection.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R532.21

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