弓形虫感染诱导小胶质细胞的活化以及米诺环素的抑制作用
发布时间：2019-03-24 16:40
【摘要】：背景及目的弓形虫是一种专性细胞内寄生原虫，能感染包括人在内的几乎所有的恒温动物。无论是先天性还是获得性感染，都对人类的健康产生严重的威胁。先天性弓形虫病主要表现为脑积水、脑钙化、出生后智力发育迟缓、癫痫等；获得性弓形虫病可表现为头痛、发热、淋巴结肿大，免疫力低下患者可并发全身严重弓形虫病，例如弓形虫脑炎等。在机体免疫缺陷或功能低下时，脑部的包囊破裂，缓殖子向速殖子转化，弓形虫隐性感染状态被活化，导致致命性弓形虫脑炎的发生。弓形虫不同的基因型与疾病的发生、发展以及结局有关。在北美和欧洲，主要的弓形虫基因型为Type I、Type II和Type III,而我国的优势基因型为Chinese1型。小胶质细胞，是中枢神经系统中主要的固有免疫成分，包括静息和活化两种状态，在脑部病理损伤时发挥着重要的作用。在感染、损伤等刺激时，小胶质细胞能活化，分化、增殖并分泌大量的神经毒性因子，发挥免疫保护功能，但是小胶质细胞过度的活化也会对神经元等周围组织产生损伤。在弓形虫感染中，对于小胶质细胞的影响尚不十分清楚。本文主要研究小胶质细胞（BV-2）细胞系在弓形虫TgCtwh6虫株（Chinese1基因型，成囊株）感染中的变化，并加入了小胶质细胞的抑制剂米诺环素(minocycline)，以了解其对小胶质细胞的抑制作用，进而为弓形虫脑病的防治提供实验基础。 方法在transwell共培养体系中用弓形虫TgCtwh6虫株感染BV-2细胞，同时用米诺环素进行干预，分为普通培养基对照组、弓形虫TgCtwh6虫株感染的代谢物作用组和米诺环素抑制剂组。在倒置显微镜下观察细胞的形态；用流式细胞术检测细胞凋亡情况；ELISA法检测细胞上清中IL-1β、IL-6、TNF-α的含量；用Griess法检测细胞上清中NO的含量；RT-PCR法检测BV-2细胞IL-1β、IL-6、TNF-α、iNOS mRNA的表达；Western blotting检测BV-2细胞iNOS蛋白的表达。 结果与对照组相比，弓形虫代谢物作用组的小胶质细胞胞体变圆，突起减少；流式结果显示BV-2细胞感染弓形虫24h且BV-2细胞与弓形虫速殖子比例为1:1时出现凋亡，培养48h,BV-2细胞与弓形虫速殖子比例为1:5时BV-2细胞的凋亡率达35.72%；弓形虫代谢物作用组的BV-2细胞分泌炎症因子，其中培养上清中IL-1β、IL-6、TNF-α、NO的含量、BV-2细胞IL-1β、IL-6、TNF-α、iNOS mRNA的表达以及iNOS蛋白的表达均随着培养时间的延长和弓形虫速殖子与BV-2细胞比例的提高逐渐增加，米诺环素能抑制上述炎症因子的产生。 结论我国流行的弓形虫Chinese1基因型TgCtwh6成囊株的代谢物作用的BV-2细胞能分泌IL-1β、IL-6、TNF-α等炎症因子，，其在对抗弓形虫的过程中可能发挥一定的作用，但是对神经元细胞也有一定的损伤；米诺环素可以抑制炎症因子的产生，提示该药对弓形虫脑病具有潜在的治疗价值。但是弓形虫TgCtwh6诱导小胶质细胞分泌炎症因子以及米诺环素抑制的机制尚待进一步研究。
[Abstract]:Background and objective Toxoplasma gondii is a specific intracellular parasite that infects almost all thermostat animals, including humans. Both congenital and acquired infections pose a serious threat to human health. The main manifestations of congenital toxoplasmosis were hydrocephalus, calcification, postnatal mental retardation, epilepsy and so on. Acquired toxoplasmosis can be characterized by headache fever lymph node enlargement and severe toxoplasmosis in patients with low immunity such as toxoplasmosis encephalitis. In the case of immune deficiency or hypofunction, the cyst of the brain breaks down, and the bradyzoites transform to the Tachyzoites, and the latent infection of Toxoplasma gondii is activated, which leads to the occurrence of fatal toxoplasmosis encephalitis (Toxoplasma gondii encephalitis). Different genotypes of Toxoplasma gondii are associated with the occurrence, development and outcome of the disease. In North America and Europe, the main genotype of Toxoplasma gondii is Type I, type II and Type III, while the dominant genotype in China is Chinese1 type. Microglia are the main innate immune components in the central nervous system, including resting and activating states, which play an important role in the pathological injury of the brain. Under the stimulation of infection and injury, microglia can activate, differentiate, proliferate and secrete a large number of neurotoxic factors, which play an immune protective role. However, excessive activation of microglia can also cause damage to peripheral tissues such as neurons. The effect of Toxoplasma gondii infection on microglia remains unclear. The aim of this study was to investigate the changes of microglial cell line (BV-2) in the infection of Toxoplasma gondii TgCtwh6 strain (Chinese1 genotype, cyst-forming strain), and to add minocycline (minocycline), a microglial inhibitor, to Toxoplasma gondii. In order to understand the inhibitory effect of Toxoplasma gondii on microglial cells and provide experimental basis for the prevention and treatment of Toxoplasma gondii encephalopathy. Methods BV-2 cells were infected with Toxoplasma gondii TgCtwh6 strain in transwell co-culture system and treated with minocycline. They were divided into three groups: the control group, the metabolite group of Toxoplasma gondii TgCtwh6 strain infection and the minocycline inhibitor group. Cell morphology was observed under inverted microscope, apoptosis was detected by flow cytometry, content of IL-1 尾 and IL-6,TNF- 伪 in cell supernatant was detected by ELISA, NO content in supernatant was measured by Griess method. The expression of IL-1 尾 and IL-6,TNF- 伪, iNOS mRNA in BV-2 cells was detected by RT-PCR assay.; Western blotting was used to detect the expression of iNOS protein in BV-2 cells. Results compared with the control group, the microglia bodies in the Toxoplasma gondii metabolite-treated group became round and the protuberances decreased. The results of flow cytometry showed that BV-2 cells infected Toxoplasma gondii for 24 hours and the ratio of BV-2 cells to Toxoplasma gondii Tachyzoites was 1: 1. The ratio of BV-2 cells to Toxoplasma gondii Tachyzoites was 1: 5. The apoptosis rate of BV-2 cells was 35.72%. Toxoplasma gondii metabolite-treated BV-2 cells secrete inflammatory factors, in which the contents of IL-1 尾, IL-6,TNF- 伪 and NO in culture supernatant, IL-1 尾, IL-6,TNF- 伪 in BV-2 cells, and IL-1 尾, IL-6,TNF- 伪 in BV-2 cells. The expression of iNOS mRNA and iNOS protein increased gradually with the prolongation of culture time and the ratio of Toxoplasma gondii Tachyzoites to BV-2 cells. Minocycline inhibited the production of these inflammatory factors. Conclusion Toxoplasma gondii (Toxoplasma gondii) Chinese1 genotype TgCtwh6 cystocyst metabolite can secrete inflammatory factors such as IL-1 尾 and IL-6,TNF- 伪, which may play a certain role in the process of anti-Toxoplasma gondii, which may play an important role in anti-Toxoplasma gondii. However, it also has some damage to neuron cells. Minocycline can inhibit the production of inflammatory factors, suggesting that the drug has potential therapeutic value for Toxoplasma gondii encephalopathy. However, the mechanism of Toxoplasma gondii TgCtwh6-induced inflammatory factor secretion and minocycline inhibition in microglia remains to be further studied.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R531.8
本文编号：2446499
[Abstract]:Background and objective Toxoplasma gondii is a specific intracellular parasite that infects almost all thermostat animals, including humans. Both congenital and acquired infections pose a serious threat to human health. The main manifestations of congenital toxoplasmosis were hydrocephalus, calcification, postnatal mental retardation, epilepsy and so on. Acquired toxoplasmosis can be characterized by headache fever lymph node enlargement and severe toxoplasmosis in patients with low immunity such as toxoplasmosis encephalitis. In the case of immune deficiency or hypofunction, the cyst of the brain breaks down, and the bradyzoites transform to the Tachyzoites, and the latent infection of Toxoplasma gondii is activated, which leads to the occurrence of fatal toxoplasmosis encephalitis (Toxoplasma gondii encephalitis). Different genotypes of Toxoplasma gondii are associated with the occurrence, development and outcome of the disease. In North America and Europe, the main genotype of Toxoplasma gondii is Type I, type II and Type III, while the dominant genotype in China is Chinese1 type. Microglia are the main innate immune components in the central nervous system, including resting and activating states, which play an important role in the pathological injury of the brain. Under the stimulation of infection and injury, microglia can activate, differentiate, proliferate and secrete a large number of neurotoxic factors, which play an immune protective role. However, excessive activation of microglia can also cause damage to peripheral tissues such as neurons. The effect of Toxoplasma gondii infection on microglia remains unclear. The aim of this study was to investigate the changes of microglial cell line (BV-2) in the infection of Toxoplasma gondii TgCtwh6 strain (Chinese1 genotype, cyst-forming strain), and to add minocycline (minocycline), a microglial inhibitor, to Toxoplasma gondii. In order to understand the inhibitory effect of Toxoplasma gondii on microglial cells and provide experimental basis for the prevention and treatment of Toxoplasma gondii encephalopathy. Methods BV-2 cells were infected with Toxoplasma gondii TgCtwh6 strain in transwell co-culture system and treated with minocycline. They were divided into three groups: the control group, the metabolite group of Toxoplasma gondii TgCtwh6 strain infection and the minocycline inhibitor group. Cell morphology was observed under inverted microscope, apoptosis was detected by flow cytometry, content of IL-1 尾 and IL-6,TNF- 伪 in cell supernatant was detected by ELISA, NO content in supernatant was measured by Griess method. The expression of IL-1 尾 and IL-6,TNF- 伪, iNOS mRNA in BV-2 cells was detected by RT-PCR assay.; Western blotting was used to detect the expression of iNOS protein in BV-2 cells. Results compared with the control group, the microglia bodies in the Toxoplasma gondii metabolite-treated group became round and the protuberances decreased. The results of flow cytometry showed that BV-2 cells infected Toxoplasma gondii for 24 hours and the ratio of BV-2 cells to Toxoplasma gondii Tachyzoites was 1: 1. The ratio of BV-2 cells to Toxoplasma gondii Tachyzoites was 1: 5. The apoptosis rate of BV-2 cells was 35.72%. Toxoplasma gondii metabolite-treated BV-2 cells secrete inflammatory factors, in which the contents of IL-1 尾, IL-6,TNF- 伪 and NO in culture supernatant, IL-1 尾, IL-6,TNF- 伪 in BV-2 cells, and IL-1 尾, IL-6,TNF- 伪 in BV-2 cells. The expression of iNOS mRNA and iNOS protein increased gradually with the prolongation of culture time and the ratio of Toxoplasma gondii Tachyzoites to BV-2 cells. Minocycline inhibited the production of these inflammatory factors. Conclusion Toxoplasma gondii (Toxoplasma gondii) Chinese1 genotype TgCtwh6 cystocyst metabolite can secrete inflammatory factors such as IL-1 尾 and IL-6,TNF- 伪, which may play a certain role in the process of anti-Toxoplasma gondii, which may play an important role in anti-Toxoplasma gondii. However, it also has some damage to neuron cells. Minocycline can inhibit the production of inflammatory factors, suggesting that the drug has potential therapeutic value for Toxoplasma gondii encephalopathy. However, the mechanism of Toxoplasma gondii TgCtwh6-induced inflammatory factor secretion and minocycline inhibition in microglia remains to be further studied.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R531.8
【共引文献】
相关博士学位论文 前1条
1 周永华;弓形虫慢性感染大鼠模型的建立及其在学习记忆能力影响研究中的应用[D];南京农业大学;2010年
相关硕士学位论文 前1条
1 李冬娜;刚地弓形虫RH株速殖子在鼠星形胶质细胞体外培养的实验观察[D];苏州大学;2010年
本文编号：2446499
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