小胶质细胞极化模型的建立及其对弓形虫增殖的影响

发布时间：2018-04-29 12:05

本文选题：BV2细胞系 + 极化　；参考：《吉林农业大学》2017年硕士论文

【摘要】：刚地弓形虫(Toxoplasma gondii)简称弓形虫,宿主范围广,呈世界性分布,常引起慢性/隐性感染。小胶质细胞(Microglia)作为中枢神经系统(Central Nervous System,CNS)中固有的免疫成分,是机体防御过程中的最易受到感染的免疫细胞之一。作为骨髓来源的细胞,称为“脑巨噬细胞”,经外界刺激可活化两种不同的表型,即经典型活化巨噬细胞(CAM)和替代型活化巨噬细胞(AAM),向M1/M2表型转变的过程称为极化(Polarization)或活化。本研究中以BV2细胞系作为模型,研究弓形虫感染对小胶质细胞活化状态的影响,以及活化的小胶质细胞对于弓形虫体外增殖的影响,为进一步阐明弓形虫与机体天然免疫系统相互作用提供了有益的参考依据。本试验中利用脂多糖(LPS)和干扰素(IFN)-γ及IL-4分别刺激诱导正常的BV2细胞,在光学和电子显微镜下观察不同刺激因子诱导的小胶质细胞的形态学变化;采用半定量PCR、蛋白免疫印迹(Western blot)及流式细胞术(Flow Cytometry)方法分别检测M1/M2标记分子分别在核酸和蛋白水平上的表达。结果表明,LPS+IFN-γ体外诱导小胶质细胞后M1型相关因子在mRNA转录和蛋白水平上均显著上调表达;利用IL-4刺激后细胞中M2型相关标记分子水平显著上调表达。在体外极化模型成功建立的基础上,利用PLK株弓形虫体外感染正常的BV2细胞,不同时间点收集细胞及培养上清,检测BV2细胞M1/M2型标记分子表达的动态变化;同时,采用Griess法和精氨酸酶活性试验分别检测细胞中诱导型一氧化氮合酶(iNOS)和精氨酸酶(Arginase-1)的酶活性。进一步通过western-blot分析iNOS、Arg-1及相关信号通路JAK-STAT和NF-κB的磷酸化状态。结果表明,与对照组相比,弓形虫体外感染12 h开始,BV2细胞的M1型相关标记分子的mRNA转录水平上有明显上调表达(p0.001),iNOS活性及蛋白表达水平明显上调,并激活JAK-STAT1、NF-κB信号通路,诱导小胶质细胞向M1型方向极化;而M2型相关分子表达和精氨酸酶活性无显著变化;CAM具有抗原递呈能力强、抑制肿瘤生长、以及抗病原微生物的特性,能够抑制或杀死病原体;而AAM分泌多种抗炎细胞因子和趋化因子具有促进组织修复、重塑和血管再生等功能。因此下一步对M1和M2型小胶质细胞对弓形虫体外增殖的影响进行评价。通过免疫荧光试验和Western-blot的检测结果表明,弓形虫感染后24 h,M1型小胶质细胞中弓形虫的增殖量显著低于M0和M2型,表明M1型活化的小胶质细胞对弓形虫增殖有明显的抑制作用。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) has a wide host range and worldwide distribution, and often causes chronic / recessive infection. Microglia, as an inherent immune component in central nervous system (CNS), is one of the most susceptible immune cells in the process of defense. The cells derived from bone marrow, called "brain macrophages", can activate two different phenotypes by external stimulation, that is, classical activated macrophages (CAM) and alternative activated macrophages (AAMN). The process of phenotypic transformation to M1/M2 is called Polarization) or activation. The effect of Toxoplasma gondii infection on the activation of microglia and the effect of activated microglia on the proliferation of Toxoplasma gondii in vitro were studied using BV2 cell line as a model. It provides a useful reference for further elucidating the interaction between Toxoplasma gondii and innate immune system. In this experiment, normal BV2 cells were stimulated by lipopolysaccharide (LPS), interferon IFN- 纬 and IL-4, respectively. The morphological changes of microglia induced by different stimulators were observed under optical and electron microscopy. The expression of M1/M2 labeled molecules at nucleic acid and protein levels was detected by semi-quantitative PCR, Western blotanalysis and flow Cytometry respectively. The results showed that the expression of M1 type related factors in microglia induced by LPS-IFN- 纬 was significantly up-regulated at the level of mRNA transcription and protein, and the expression of M2 type related marker molecules in the cells stimulated by IL-4 was significantly up-regulated. On the basis of successful establishment of polarization model in vitro, PLK strain Toxoplasma gondii was used to infect normal BV2 cells in vitro. The cells and supernatants were collected at different time points to detect the dynamic changes of M1/M2 marker molecules expression in BV2 cells. The activities of inducible nitric oxide synthase (iNOS) and arginase-1 (Arginase-1) were detected by Griess assay and arginase activity test, respectively. The phosphorylation of iNOS Arg-1 and related signaling pathways JAK-STAT and NF- 魏 B was further analyzed by western-blot. The results showed that compared with the control group, the mRNA transcription level of M1 related marker molecules in Toxoplasma gondii infected BV2 cells increased significantly at 12 h after infection in vitro, and the activity and protein expression of p0.001Toxoplasma gondii iNOS were up-regulated, and JAK-STAT1 was activated by NF- 魏 B signaling pathway. The microglia were induced to polarize to M1 type, but the expression of M2 related molecules and arginase activity did not change significantly. CAM had strong ability of presenting antigen, inhibiting tumor growth, and anti-pathogenic microorganism, which could inhibit or kill pathogens. The secretion of many anti-inflammatory cytokines and chemokines by AAM can promote tissue repair, remodeling and vascular regeneration. Therefore, the effects of M 1 and M 2 microglia on the proliferation of Toxoplasma gondii in vitro were evaluated. The results of immunofluorescence test and Western-blot test showed that the proliferation of Toxoplasma gondii M 1 microglia was significantly lower than that of M 0 and M 2 microglia at 24 h after infection, indicating that M1 activated microglia could inhibit the proliferation of Toxoplasma gondii.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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