【作者】 周敏；

【导师】 杨勇军；

【作者基本信息】 吉林大学 ， 预防兽医学， 2015， 硕士

【摘要】 单增李斯特菌是一种常见的革兰氏阳性胞内寄生菌。虽然单增李斯特菌感染的发生率不高，但感染后果严重，给人类健康和畜牧业生产带来严重危害。单增李斯特菌极易感染老人、孕妇以及新生儿等免疫力低下的人群。妊娠妇女在感染该菌后，会出现流产、早产、胎儿发育障碍等临床症状。天然免疫系统是宿主识别和抵御病原入侵的第一道防线，而炎性体是天然免疫系统的重要组成部分。炎性体是一种由模式识别受体、pro-caspase-1和接头蛋白ASC共同形成的胞浆内的多蛋白复合物。炎性体装配形成后，促进caspase-1活化，从而导致促炎性细胞因子IL-1β和IL-18的剪切和分泌。另外，炎性体活化能够促进细胞发生一种依赖于caspase-1活化的细胞程序性死亡，这种死亡方式被定义为“pyroptosis”。炎性体能被多种病原和病原的成分活化，包括单增李斯特菌、尼日利亚菌素、病原DNA等。而炎性体活化对于抵抗和清除入侵的病原具有重要意义。到目前为止，单增李斯特菌活化母胎界面胎盘滋养细胞炎性体信号的研究尚未见报道。胎盘是母体和胎儿相互连接的桥梁和纽带，也是母胎之间物质能量交换重要场所。胎盘滋养细胞参与胎盘屏障的形成，能阻止病原等有害物质的威胁。滋养细胞不仅具有吞噬功能，还表达多种天然免疫模式识别受体（PRRs），能够针对病原感染作出相应的免疫反应，以抵御病原的入侵。因此，我们猜测胎盘滋养细胞可能存在功能性的炎性体并在抵御单增李斯特菌入侵中发挥重要作用。本课题首先利用单增李斯特菌感染妊娠小鼠，检测胎盘炎性体活化情况。结果显示，单增李斯特菌感染孕鼠后，胎盘组织caspase-1及IL-1β活化片段表达明显增多，而且IL-1β的分泌水平也显著提高，表明单增李斯特菌感染的孕鼠胎盘存在炎性体活化。滋养细胞作为胎盘的主要构成细胞，对于胎盘发挥屏障功能具有重要作用。因此，为了确定胎盘组织炎性体的活化是由胎盘滋养细胞所介导的，我们接着以滋养细胞为主要研究对象，进行了相应研究。首先，运用PCR、Western Blot和免疫组化的方法，分析了人胎盘绒毛组织和多种滋养细胞系上NLRP3和AIM2炎性体相关基因的表达情况。结果发现，HTR-8/SV40、JAR、JEG3三种滋养细胞系和人胎盘绒毛组织均表达炎性体相关基因NLRP3、AIM2、ASC和caspase-1，这些结果表明滋养细胞上可能存在功能性的炎性体。紧接着，利用LPS联合单增李斯特菌感染HTR-8/SV40细胞，进而分析炎性体的活化。我们发现，IL-1β的裂解片段表达上调，caspase-1酶活性增强，IL-1β分泌显著增多，这与在体实验一致的结果，由此确定单增李斯特菌感染的胎盘炎性体的活化确实是滋养细胞所引起的。与此同时，我们还发现HTR-8细胞感染单增李斯特菌后，NLRP3蛋白水平显著上调，提示滋养细胞NLRP3炎性体可能被单增李斯特菌激活。接下来，我们分离了C57BL/6J和NLRP3-/-小鼠来源的原代滋养细胞。在感染单增李斯特菌后，ELISA检测IL-1β的分泌，发现NLRP3敲除后，IL-1β分泌减少。这一结果证实了单增李斯特菌能活化滋养细胞的NLRP3炎性体。最后，我们利用单增李斯特菌分别感染C57BL/6J和NLRP3-/-孕鼠，比较妊娠结局。结果发现，NLRP3敲除后，小鼠流产严重，流产率上升。综上所述，本课题证明了滋养细胞通过活化NLRP3炎性体，能有效阻止单增李斯特菌诱导的流产，这让我们对胎盘滋养细胞抵御病原感染的分子机制有了新的认识。更多还原

【Abstract】 Listeria monocytogenes is a Gram-positive intracellular bacterium whichubiquitously exsits. Although the incidence of listeriosis is lower, Listeriamonocytogenes pose a great threat to human health and agriculture because of thesevere effects. Listeria monocytogenes especially infect immunosuppressedindividuals, such as the elder, the neonatus and pregnant women and inducepregnancy complications, such as miscarry, preterm labor, fetal developmentaldisability.Innate immune system act as the primary defense against invading pathogenicmicrobes. As a part of innate immune system, inflammasome is a large cytoplasmiccomplexes which consist of PRRs, pro-caspase-1and the adaptor ASC. With theformation of inflammasome, it catalyses the auto-proteolytic activation ofpro-caspase-1which results in IL-1β/IL-18maturation and cell death termed“pyrotosis”, a type of programmed cell death depending on the activation ofcaspase-1. Pathogenic microbes or its components, such as Listeria monocytogene,nigericin, microbial DNA can trigger inflammasome’s activation which plays a rolein the battle adainst invading microbes. Up to now, it have not been reported thatListeria monocytigenens active trophoblastic inflammasome in the maternal-fetalinterface.Not only is the placenta a bridge linking mother and fetus, but also a placewhere they can exchange mutual matter and energe. Trophoblast cells are part ofplacental barrier which prevent pathogens crossing over. Moreover, trophoblast cellsexhibit intense phagocytic activity and express many PRRs, which make trophoblastcells respond to invading pathogen. Therefore,wo hypothesize that functionalinflammasomes could exist in trophoblast cells and probably play a role in the defence against microbes.This research was conducted to primarily detect the activation of inflammasomein Listeria-infected placenta. The results showed that bioactive caspase-1and IL-1βwere elevated and the secretion of IL-1β was increased, which gave an evidence tosupport the activation of inflammasome in placenta.Trophoblast cells are part of placental barrier and are very important forplacental barrier. In order to confirm the activation of inflammasome in placenta isdue to the trophoblast cells, we explore the expression of NLRP3inflammasome andAIM2inflammasome associated genes in human placental villi and varioustrophoblast cell lines by PCR, Western Blot and immunohistochemical. And wefound that human placental villi and3trophoblast cell lines: HTR-8/SV40, JAR,JEG3, all of them constitutively expressed inflammasome associated genes such asNLRP3, AIM2, ASC, caspase-1. This implied trophoblast cells could functionallyactive inflammasome. Then we analyzed the activation of HTR-8/SV40infectedwith Listeria monocytogenes in the presence of LPS or not. We found that bioactiveIL-1β was increased, proteolytic activitity of caspase-1was enhanced and thesecretion of IL-1βrose. These results were consistant with that in vivo and weensured the activation of inflammasome in trophoblast cells contributed to that inListeria-infected placenta. Furthermore, we observed an increased NLRP3whichindicated the activation of NLRP3inflammasome. Subsequently, wo isolatedprimary trophoblast cells derived from C57BL/6J and NLRP3-/-, following by theinfection with Listeria monocytogenes. The results showed an attenuated secretion ofIL-1β and confirmed our assumption.Finally, we infected pregnant C57BL/6J and NLRP3-/-mice with Listeriamonocytogenes respectively. The results showed an more detrimental pregnancyoutcome in NLRP3-/-mice and an increased fetal loss rate in comparison withC57BL/6J mice.In conclusion, this research revealed an function role of trophoblast cells whichcould prevent Listeria-induced abortion via NLRP3inflammasome and provided an insight into the molecular mechanism of the role of trophoblast cells in thedefence against microbes.更多还原