本文選題：微小隱孢子蟲 + TLRs��； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：微小隱孢子蟲是一種常見(jiàn)的頂復(fù)門原蟲，常寄生在腸道上皮細(xì)胞中，可通過(guò)污染水源而造成隱孢子蟲病的爆發(fā)，同時(shí)，微小隱孢子蟲的感染也是器官移植、艾滋病患者等處于免疫抑制狀態(tài)病人的致死因素之一。目前，市場(chǎng)上尚未有有效的防治隱孢子蟲病的藥物和疫苗，微小隱孢子蟲感染宿主細(xì)胞后，宿主細(xì)胞的抗感染機(jī)制中諸多細(xì)節(jié)也還不清楚。為探索隱孢子蟲病的有效防控手段，闡明宿主-病原相互作用機(jī)制顯得尤為重要。研究表明，宿主細(xì)胞Toll樣受體（Toll-likereceptors，TLRs）在對(duì)寄生蟲的識(shí)別和相互作用中起著關(guān)鍵作用，宿主細(xì)胞在寄生蟲刺激后，激活對(duì)應(yīng)的TLRs和下游NF-κB信號(hào)通路，并使相應(yīng)的細(xì)胞因子分泌量增加。然而，作為一種主要寄生在腸道，主要感染小腸上皮細(xì)胞的寄生性原蟲，人小腸上皮細(xì)胞TLRs對(duì)微小隱孢子蟲的識(shí)別機(jī)制卻未見(jiàn)報(bào)道，微小隱孢子蟲感染體外培養(yǎng)的小鼠細(xì)胞后TLRs的表達(dá)情況也不清楚。本研究旨在研究當(dāng)感染了微小隱孢子蟲后，宿主細(xì)胞TLRs對(duì)微小隱孢子蟲的識(shí)別情況。 微小隱孢子蟲刺激Caco-2和RAW264.7細(xì)胞TLRs激活情況。在微小隱孢子蟲子孢子刺激細(xì)胞2h后熒光定量PCR檢測(cè)TLRs的表達(dá)情況，2h后Westernblot檢測(cè)NF-κB激活情況，12h后ELISA檢測(cè)TNF-α的分泌情況。試驗(yàn)結(jié)果表明當(dāng)Caco-2細(xì)胞感染了微小隱孢子蟲后，TLR2和TLR4得到顯著的激活并引起下游NF-κB信號(hào)通路的激活和TNF-α的分泌，而當(dāng)RAW264.7細(xì)胞感染了微小隱孢子蟲后，檢測(cè)到TLR2、TLR4、TLR11和TLR12的激活以及下游NF-κB信號(hào)通路的激活和TNF-α的分泌。 微小隱孢子蟲分泌性抗原刺激RAW264.7細(xì)胞TLRs的激活情況。為探明能引起TLR11和TLR12激活的分子是否來(lái)自于分泌性抗原，分別采用微小隱孢子蟲子孢子超聲裂解抗原以及分泌性抗原刺激RAW264.7細(xì)胞，檢測(cè)TLRs的表達(dá)情況、NF-κB激活情況以及TNF-α的分泌情況。試驗(yàn)結(jié)果顯示，微小隱孢子蟲子孢子超聲裂解抗原能夠引起TLR2、TLR4、TLR11和TLR12的激活，而分泌性抗原僅能引起TLR11的激活，微小隱孢子蟲子孢子超聲裂解抗原以及分泌性抗原刺激RAW264.7細(xì)胞后均能引起NF-κB激活以及TNF-α的分泌。由此確定微小隱孢子蟲子孢子分泌性抗原中確實(shí)存在著能激活TLR11的成分，能夠激活TLR12的成分則不存在于分泌性抗原中而是存在于微小隱孢子蟲子孢子超聲裂解抗原中。弓形蟲分泌性抗原中能夠激活TLR11和TLR12的分子是弓形蟲profilin蛋白，通過(guò)對(duì)已完成的微小隱孢子蟲基因組的查詢，發(fā)現(xiàn)微小隱孢子蟲也存在著profilin蛋白且其與弓形蟲profilin蛋白具有較高的同源性（63%），微小隱孢子蟲profilin（CpProfilin）也是一種分泌性抗原分子。 CpProfilin刺激RAW264.7細(xì)胞TLRs的表達(dá)情況。為進(jìn)一步驗(yàn)證微小隱孢子蟲分泌性抗原profilin蛋白能夠激活TLR11而不引起TLR12的激活，本研究通過(guò)構(gòu)建CpProfilin原核表達(dá)體系，表達(dá)并純化CpProfilin蛋白，在CpProfilin刺激RAW264.7細(xì)胞后檢測(cè)TLRs的表達(dá)情況、NF-κB激活情況以及TNF-α的分泌情況。此外，構(gòu)建轉(zhuǎn)染了TLR11的HEK293T細(xì)胞，，在CpProfilin刺激細(xì)胞后檢測(cè)IL-12的分泌情況。試驗(yàn)結(jié)果顯示，CpProfilin能夠激活TLR11而不激活其他TLRs，能夠引起NF-κB信號(hào)轉(zhuǎn)導(dǎo)通路的激活以及TNF-α的分泌量增加，CpProfilin刺激轉(zhuǎn)染了TLR11的HEK293T細(xì)胞后檢測(cè)到IL-12的表達(dá)量上調(diào)。 綜上所述，Caco-2細(xì)胞TLR2和TLR4參與了對(duì)微小隱孢子蟲的識(shí)別；RAW264.7細(xì)胞TLR2、TLR4、TLR11和TLR12參與了對(duì)微小隱孢子蟲的識(shí)別；微小隱孢子蟲分泌性抗原中的CpProfilin能夠被TLR11特異性的識(shí)別而不被其他TLRs所識(shí)別。
[Abstract]:Cryptosporidium parvum is a common protozoa, often parasitic in intestinal epithelial cells, and can cause the outbreak of cryptosporidiosis by polluting the water source. At the same time, the infection of the small Cryptosporidium is also one of the fatal factors of the patients in the immunosuppressive state, such as organ transplantation and AIDS patients. The market has not yet been effective. Drugs and vaccines for the control of Cryptosporidium, after infecting host cells of Cryptosporidium parvum, many details of the host cell's anti infection mechanism are still unclear. It is particularly important to explore the effective means of prevention and control of Cryptosporidium and elucidate the host pathogen interaction mechanism. The study shows that the host cell Toll like receptor (Toll-likereceptor) S, TLRs) plays a key role in the identification and interaction of parasites. The host cells activate the corresponding TLRs and downstream NF- kappa B signaling pathway after the parasite stimulation, and increase the secretion of the corresponding cytokines. However, as a parasitic protozoa mainly parasitic in the intestines, mainly infected small intestinal epithelial cells, human small intestinal epithelium. The identification mechanism of cell TLRs for Cryptosporidium was not reported. The expression of TLRs in mouse cells cultured in vitro was not clear. The purpose of this study was to study the identification of TLRs for Cryptosporidium parvum in the host cells.
The activation of TLRs in Caco-2 and RAW264.7 cells was stimulated by Cryptosporidium parvum. The expression of TLRs was detected by fluorescence quantitative PCR after 2h of Cryptosporidium spore of small Cryptosporidium. 2h after 2H was used to detect the activation of NF- kappa B. 2 and TLR4 were significantly activated and resulted in the activation of the downstream NF- kappa B signaling pathway and the secretion of TNF- alpha. When RAW264.7 cells infected with Cryptosporidium tiny, the activation of TLR2, TLR4, TLR11 and TLR12, and the activation of the downstream NF- kappa B signaling pathway and the secretion of TNF- alpha were detected.
The activation of RAW264.7 cell TLRs was stimulated by secretory antigen of Cryptosporidium parvum. It was found that whether the molecules activated by TLR11 and TLR12 were derived from secretory antigens, using ultrasonic lysis antigen of Cryptosporidium spore and secretory antigen to stimulate RAW264.7 cells respectively, to detect the expression of TLRs, the activation of NF- kappa B and the activation of NF- kappa B, as well as the activation of NF- kappa B. The results of the secretion of TNF- alpha showed that the ultrasonic lysis antigen of the sporozoites of the small Cryptosporidium could cause activation of TLR2, TLR4, TLR11 and TLR12, and the secretory antigen could only cause the activation of TLR11, and the activation of NF- kappa B and TNF- could cause NF- kappa B activation and TNF- after the secretory antigen was stimulated by the secretory antigen of RAW264.7 cells. It is determined that the secretory antigen of the sporozoite of small Cryptosporidium does exist to activate the TLR11, and that the activation of TLR12 is not in the secretory antigen but in the ultrasonic lysis antigen of the sporozoite of the small Cryptosporidium. The molecules that can activate TLR11 and TLR12 in the secretory antigenic of Toxoplasma gondii are the arches Profilin protein, through the search for the genome of the completed Cryptosporidium, found that the profilin protein also existed in the Cryptosporidium parvum and had high homology with the Toxoplasma gondii profilin protein (63%), and the profilin (CpProfilin) of Wei Xiaoyin Sporozoa (CpProfilin) was also a secretory antigen.
CpProfilin stimulates the expression of TLRs in RAW264.7 cells. In order to further verify that the secretory antigen of Cryptosporidium parvum can activate TLR11 without activating the activation of TLR12, this study expresses and purifies CpProfilin protein by constructing the CpProfilin prokaryotic expression system, and detects TLRs expression after CpProfilin stimulates RAW264.7 cells. Moreover, the activation of NF- kappa B and the secretion of TNF- alpha. In addition, the HEK293T cells transfected with TLR11 were constructed and the secretion of IL-12 was detected after CpProfilin stimulation. The results showed that CpProfilin could activate TLR11 without activating other TLRs, which could cause activation of the NF- kappa B signal transduction pathway and the increase of the secretion of alpha. Filin stimulated the transfection of TLR11 HEK293T cells and detected the up regulation of IL-12 expression.
To sum up, Caco-2 cells TLR2 and TLR4 participated in the identification of Cryptosporidium parvum; RAW264.7 cells TLR2, TLR4, TLR11 and TLR12 participated in the identification of Cryptosporidium parvum; CpProfilin in the secretory antigen of Cryptosporidium can be identified by TLR11 specificity without being identified by other TLRs.

【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】：2015
【分類號(hào)】：S852.723

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