微小隐孢子虫刺激对宿主细胞TLRs的影响

发布时间：2018-05-05 22:22

本文选题：微小隐孢子虫 + TLRs　；参考：《吉林大学》2015年硕士论文

【摘要】：微小隐孢子虫是一种常见的顶复门原虫，常寄生在肠道上皮细胞中，可通过污染水源而造成隐孢子虫病的爆发，同时，微小隐孢子虫的感染也是器官移植、艾滋病患者等处于免疫抑制状态病人的致死因素之一。目前，市场上尚未有有效的防治隐孢子虫病的药物和疫苗，微小隐孢子虫感染宿主细胞后，宿主细胞的抗感染机制中诸多细节也还不清楚。为探索隐孢子虫病的有效防控手段，阐明宿主-病原相互作用机制显得尤为重要。研究表明，宿主细胞Toll样受体（Toll-likereceptors，TLRs）在对寄生虫的识别和相互作用中起着关键作用，宿主细胞在寄生虫刺激后，激活对应的TLRs和下游NF-κB信号通路，并使相应的细胞因子分泌量增加。然而，作为一种主要寄生在肠道，主要感染小肠上皮细胞的寄生性原虫，人小肠上皮细胞TLRs对微小隐孢子虫的识别机制却未见报道，微小隐孢子虫感染体外培养的小鼠细胞后TLRs的表达情况也不清楚。本研究旨在研究当感染了微小隐孢子虫后，宿主细胞TLRs对微小隐孢子虫的识别情况。微小隐孢子虫刺激Caco-2和RAW264.7细胞TLRs激活情况。在微小隐孢子虫子孢子刺激细胞2h后荧光定量PCR检测TLRs的表达情况，2h后Westernblot检测NF-κB激活情况，12h后ELISA检测TNF-α的分泌情况。试验结果表明当Caco-2细胞感染了微小隐孢子虫后，TLR2和TLR4得到显著的激活并引起下游NF-κB信号通路的激活和TNF-α的分泌，而当RAW264.7细胞感染了微小隐孢子虫后，检测到TLR2、TLR4、TLR11和TLR12的激活以及下游NF-κB信号通路的激活和TNF-α的分泌。微小隐孢子虫分泌性抗原刺激RAW264.7细胞TLRs的激活情况。为探明能引起TLR11和TLR12激活的分子是否来自于分泌性抗原，分别采用微小隐孢子虫子孢子超声裂解抗原以及分泌性抗原刺激RAW264.7细胞，检测TLRs的表达情况、NF-κB激活情况以及TNF-α的分泌情况。试验结果显示，微小隐孢子虫子孢子超声裂解抗原能够引起TLR2、TLR4、TLR11和TLR12的激活，而分泌性抗原仅能引起TLR11的激活，微小隐孢子虫子孢子超声裂解抗原以及分泌性抗原刺激RAW264.7细胞后均能引起NF-κB激活以及TNF-α的分泌。由此确定微小隐孢子虫子孢子分泌性抗原中确实存在着能激活TLR11的成分，能够激活TLR12的成分则不存在于分泌性抗原中而是存在于微小隐孢子虫子孢子超声裂解抗原中。弓形虫分泌性抗原中能够激活TLR11和TLR12的分子是弓形虫profilin蛋白，通过对已完成的微小隐孢子虫基因组的查询，发现微小隐孢子虫也存在着profilin蛋白且其与弓形虫profilin蛋白具有较高的同源性（63%），微小隐孢子虫profilin（CpProfilin）也是一种分泌性抗原分子。 CpProfilin刺激RAW264.7细胞TLRs的表达情况。为进一步验证微小隐孢子虫分泌性抗原profilin蛋白能够激活TLR11而不引起TLR12的激活，本研究通过构建CpProfilin原核表达体系，表达并纯化CpProfilin蛋白，在CpProfilin刺激RAW264.7细胞后检测TLRs的表达情况、NF-κB激活情况以及TNF-α的分泌情况。此外，构建转染了TLR11的HEK293T细胞，，在CpProfilin刺激细胞后检测IL-12的分泌情况。试验结果显示，CpProfilin能够激活TLR11而不激活其他TLRs，能够引起NF-κB信号转导通路的激活以及TNF-α的分泌量增加，CpProfilin刺激转染了TLR11的HEK293T细胞后检测到IL-12的表达量上调。综上所述，Caco-2细胞TLR2和TLR4参与了对微小隐孢子虫的识别；RAW264.7细胞TLR2、TLR4、TLR11和TLR12参与了对微小隐孢子虫的识别；微小隐孢子虫分泌性抗原中的CpProfilin能够被TLR11特异性的识别而不被其他TLRs所识别。
[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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.723

【相似文献】