本文關(guān)鍵詞： 旋毛蟲成蟲 絲氨酸蛋白酶 免疫細(xì)胞 細(xì)胞毒性 增殖性應(yīng)答　出處：《吉林大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：旋毛蟲病是由于人和動(dòng)物生食或半生食含有旋毛蟲感染性L1幼蟲的肉或肉制品而引發(fā)的一種人獸共患病。本病致死率雖然不高（1-2%），僅重度感染才能造成嚴(yán)重的心肌及大腦損傷而導(dǎo)致死亡，但是在其腸道侵襲期通過免疫調(diào)控抑制宿主免疫力，同時(shí)干擾了各種細(xì)菌、病毒以及其他寄生蟲疫苗的免疫預(yù)防效果，大大提高了其他病原體入侵宿主的可能性，對(duì)人類身體健康構(gòu)成了巨大的威脅。但是目前對(duì)于這些旋毛蟲調(diào)節(jié)宿主免疫反應(yīng)的毒力分子不確定性及其作用機(jī)制尚不清楚導(dǎo)致了有關(guān)旋毛蟲病的預(yù)防制劑的研發(fā)始終沒有突破性的進(jìn)展。本實(shí)驗(yàn)室通過構(gòu)建了旋毛蟲3日齡成蟲cDNA文庫，，并利用免疫學(xué)篩選獲得了高豐度和強(qiáng)抗原性的絲氨酸蛋白酶基因Zh68。寄生蟲絲氨酸蛋白酶在宿主與寄生蟲之間互相作用過程（如入侵宿主組織、寄生蟲營養(yǎng)的攝取和逃避宿主免疫應(yīng)答）中發(fā)揮了重要作用，甚至有研究表明絲氨酸蛋白酶可作為多種寄生蟲感染性疾病的診斷標(biāo)志。 本實(shí)驗(yàn)通過對(duì)旋毛蟲成蟲腸道感染期宿主多種免疫細(xì)胞的動(dòng)態(tài)變化監(jiān)測來初步剖析宿主免疫抑制的變化規(guī)律。首先將500條L1幼蟲口服感染昆明小鼠，發(fā)現(xiàn)在感染后第1天小鼠腸道成蟲回收率為65%，直至感染后第17天小鼠腸道成蟲完全排出；感染后第5天到第9天是成蟲穩(wěn)定的腸道感染期，此時(shí)雌蟲大量釋放新生幼蟲，并于第7天達(dá)到高峰。隨后的流式細(xì)胞術(shù)檢測結(jié)果顯示感染后第7天CD4~+T淋巴細(xì)胞數(shù)量大量減少，同時(shí)T淋巴細(xì)胞對(duì)刀豆素A（Con A）的增殖性應(yīng)答能力降低。本實(shí)驗(yàn)室在前期構(gòu)建的新生幼蟲cDNA文庫中也篩選到了特異性絲氨酸蛋白酶基因T668，因此很有可能是成蟲絲氨酸蛋白酶Zh68和新生幼蟲特異性絲氨酸蛋白酶T668共同導(dǎo)致了這一結(jié)果的產(chǎn)生。在旋毛蟲感染后的前7天，T淋巴細(xì)胞增多，以CD8~+T淋巴細(xì)胞為主，導(dǎo)致CD4~+/CD8~+比值下降，表明小鼠免疫力降低。從旋毛蟲感染后第11天開始，CD4~+T細(xì)胞明顯增多，小鼠免疫力逐步恢復(fù)。同時(shí)發(fā)現(xiàn)在旋毛蟲腸道感染前期（感染前7天），小鼠巨噬細(xì)胞數(shù)量急劇下降，相對(duì)于LPS的刺激，B淋巴細(xì)胞對(duì)旋毛蟲抗原無增殖性應(yīng)答；從感染后第11天開始巨噬細(xì)胞數(shù)量恢復(fù)，B淋巴細(xì)胞數(shù)量開始上升。 鑒于旋毛蟲腸道感染前期小鼠巨噬細(xì)胞急劇性變化，我們進(jìn)一步通過體外實(shí)驗(yàn)研究旋毛蟲成蟲排泄分泌產(chǎn)物（ES產(chǎn)物）中的絲氨酸蛋白酶對(duì)巨噬細(xì)胞S774A.1的作用機(jī)制。CCK-8的實(shí)驗(yàn)結(jié)果表明成蟲ES產(chǎn)物以劑量依賴性和時(shí)間依賴性的方式對(duì)S774A.1巨噬細(xì)胞產(chǎn)生毒性作用，但用抗成蟲絲氨酸蛋白酶Zh68的抗體封閉ES產(chǎn)物之后，這一毒性作用大大減少；通過Annexin V/PI雙標(biāo)記流式細(xì)胞術(shù)檢測結(jié)果表明成蟲絲氨酸蛋白酶Zh68可以誘導(dǎo)巨噬細(xì)胞S774A.1凋亡。 目前有關(guān)旋毛蟲免疫逃逸相關(guān)基因的研究薄弱，研究旋毛蟲絲氨酸蛋白酶在蟲體侵襲及寄生過程中的功能及作用靶標(biāo)、將為闡明旋毛蟲侵襲過程中的免疫逃逸機(jī)制以及新型的抗旋毛蟲及相關(guān)寄生蟲藥物研發(fā)打下結(jié)實(shí)基礎(chǔ)。
[Abstract]:Trichinellosis is a zoonosis caused by human and animal eating raw or undercooked food containing L1 larvae infected with Trichinella spiralis meat or meat products. The prevalence of this disease mortality rate is not high (1-2%), only severe infections can cause serious heart and brain damage and lead to death, but is in theintestinal phase through the inhibition of immune regulation of host immunity, and the interference of a variety of bacteria, viruses and other parasites vaccine immune prevention effect, greatly improving the possibility of other pathogens invade the host, poses a great threat to human health. But at present for the virulence genes of Trichinella spiralis modulate the host immune response uncertainty and its mechanism of action is not clear cause the research on the prevention of trichinosis has not a breakthrough. The laboratory was constructed by Trichinella spiralis 3 days old adult cDNA library, And the use of immunological screening to obtain the high abundance and high antigenicity of the serine protease gene Zh68. parasites between serine proteases in the host parasite interaction process (such as the invasion of host tissues, the uptake of nutrients of parasites and evade the host immune response) play an important role, and even research indicates that serine protease can be used for diagnosis of infectious diseases of various parasites mark.
Through this experiment, dynamic monitoring of host immune cells of adult Trichinella spiralis intestinal infection to the preliminary analysis of changes of host immune suppression. The 500 L1 larvae of oral infection in Kunming mice, found in first days after infection of mice intestinal adult worm recovery rate is 65%, until seventeenth days after infection of mouse intestinal adult infection completely discharged; after fifth to ninth days is a stable adult intestinal infection period, the release of a large number of female newborn larvae, peaked at seventh day. The results of flow cytometry showed that seventh days after infection the number of lymph cells reduce subsequent CD4~+T, while T lymphocytes to concanavalin A (Con A) reduced the proliferative response ability of newborn larvae cDNA Library in our laboratory previously constructed also screened specific serine protease gene T668, so it's possible that the adult serine protease Zh6 8 newborn larvae and specific serine protease T668 leads to the results. In the first 7 days after infection with Trichinella spiralis, T lymphocytes increased to CD8~+T lymphocytes, resulting in the decrease of the ratio of CD4~+/CD8~+, showed that the immune system of mice decreased. From the eleventh day after infection with Trichinella spiralis, CD4~+T cells significantly increased the immunity of mice gradually restored. At the same time found in the early stage of intestinal infection of Trichinella spiralis (7 days before infection), a sharp decline in the number of macrophages of mice, compared with LPS stimulation, B lymphocyte proliferative response to antigen of Trichinella spiralis infection after eleventh days; the number of macrophages began recovery, the number of B lymphocytes began to rise.
In view of early intestinal infection of Trichinella spiralis in mouse macrophages change dramatically, we further product in vitro study of adult Trichinella spiralis excretory secretory (ES product) mechanism of.CCK-8 on macrophage S774A.1 serine protease in experimental results show that the adult ES product in a dose-dependent and time-dependent manner have toxic effects on S774A.1 macrophages, but after with the antibody against adult serine protease Zh68 closed ES products, which greatly reduce the toxicity; by Annexin V/PI double labeled flow cytometry results showed that adult serine protease Zh68 can induce apoptosis of S774A.1 macrophages.
At present the researches on immune escape related gene of Trichinella spiralis is weak, function and role in parasite invasion and parasitic in the process of target of Trichinella spiralis serine protease, will escape mechanism and new anti Trichinella parasites and lay a solid foundation for drug development in the process of invasion of Trichinella spiralis immune to clarify.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【共引文獻(xiàn)】

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