NK细胞调控LPS诱导的神经系统炎症

发布时间：2018-07-04 20:08

本文选题：神经系统炎症 + 小胶质细胞　；参考：《泰山医学院》2014年硕士论文

【摘要】：目的本实验以腹腔注射脂多糖(Lipopolysaccharide, LPS)诱导的C57BL/6小鼠的神经系统炎症为模型，通过抗体清除外周及脑内浸润的自然杀伤细胞(Natural killer cells,NK)，检测NK细胞在神经炎症中对小胶质细胞活化和炎症细胞浸润的影响，以探讨NK细胞在LPS诱导的神经系统炎症中的作用。方法 1、动态观察和监测LPS处理不同时间点小鼠体重，绘制体重变化曲线以确定系统性炎症的变化。于LPS或PBS处理两次时同时尾静脉注射4%Evans Blue200ul，次日用1%戊巴比妥钠100ul麻醉小鼠，待其深入麻醉时用冰PBS心脏灌注，取脑观察Evans Blue是否浸润脑组织。正常情况下由于血脑屏障(Blood brain barrier，BBB)的存在，Evans Blue不能进入脑组织，若BBB的完整性遭到损伤或者BBB通透性增加时，Evans Blue会经外周血液循环浸润到脑内。以此来探讨LPS诱导的系统性炎症能否够引起BBB的破坏。 2、免疫荧光抗体标记脑单细胞后，流式分析NK细胞及其他炎症细胞向CNS的浸润，同时用免疫荧光检测脑组织小胶质细胞的活化状态，以此来探讨LPS能否引起CNS的炎症反应。 3、用抗NK抗体(PK136)清除外周NK细胞，清除NK细胞后用LPS处理三次，LPS20微克/只/天(以下称为LPS+PK136组)，并于每次LPS处理时检测小鼠体重。用流式分析CNS中浸润免疫细胞的变化，同时免疫荧光检测PBS组、LPS组和LPS+PK136组脑组织小胶质细胞Iba1的表达情况和脑组织炎性因子mRNA的表达水平。 4、为了解NK细胞在LPS诱导的神经系统炎症中的细胞学机制，动态检测LPS处理不同时间点NK细胞及其他免疫细胞向CNS的浸润，用抗NK抗体预先清除外周NK细胞后进而LPS处理，流式检测浸润的中性粒细胞和单核细胞的变化。结果 1、LPS处理后小鼠体重逐步减轻，于第二、三天降至最低，随后又呈上升趋势，故本实验采用LPS处理二天或三天炎症较重时进行检测。经4%Evans Blue处理后发现LPS组的脑组织被染料着色，而PBS组脑组织基本上没有颜色变化，说明BBB的完整性遭到破坏。此实验证明LPS诱导系统性炎症的同时也引起了BBB通透性的增加，提示外周的免疫细胞可能会浸润到CNS中。 2、在脑组织流式分析中发现LPS处理后CNS中有大量白细胞浸润，包括NK细胞、中性粒细胞和单核细胞显著增加，提示此时CNS内发生了炎症反应。脑组织免疫荧光Iba1染色发现LPS组较PBS组小胶质细胞明显活化。此外，脑组织mRNA检测发现LPS组比PBS组IL-1表达水平显著性增加。此实验说明腹腔注射LPS诱导系统性炎症的同时也引起了神经系统的炎症。 3、预先清除NK细胞后进而LPS腹腔注射，发现脑内浸润的炎症细胞较未清除NK组显著性降低，小胶质细胞活化明显减轻，体重显著性增加，IL-1表达也显著性降低。此实验提示NK细胞在LPS诱导的神经系统炎症中可能起促炎作用。 4、在LPS诱导的神经炎症模型中，，CNS中NK细胞的浸润早于中性粒细胞。预先清除NK细胞后CNS中浸润的中性粒细胞和单核细胞显著性降低。此实验说明在LPS诱导的神经炎性模型中，浸润的NK细胞可能通过吸引外周中性粒细胞和单核细胞进而促进CNS的炎症反应。结论腹腔注射LPS引起全身系统性炎症的同时也可以诱导神经系统发生炎症，伴随大量免疫细胞的浸润，清除其中的NK细胞导致神经系统的炎症减轻和脑内中性粒细胞、单核细胞浸润减少，说明NK细胞参与调控神经系统炎症，这种调控可能是NK细胞进入脑组织后进一步促进外周中性粒细胞和单核细胞向CNS浸润而实现。
[Abstract]:objective
In this experiment, a model of C57BL/6 mice induced by intraperitoneal injection of Lipopolysaccharide (LPS) was used to investigate the effects of NK cells on the activation of small colloidal cells and infiltration of inflammatory cells in neuroinflammation by using antibodies to clear the peripheral and intraperitoneal infiltration of natural killer cells (Natural killer cells, NK). The role of cell in the inflammation of the nervous system induced by LPS.
Method
1, dynamically observed and monitored the weight of LPS in mice at different time points, plotted the curve of weight change to determine the change of systemic inflammation. 4%Evans Blue200ul was injected into the tail vein at the time of LPS or PBS treatment, and the next day, the mice were anesthetized with 1% pentobarbital sodium 100ul, and the ice PBS heart was perfused to observe whether Evans Blue was in the brain. In the presence of Blood brain barrier (BBB) in normal conditions, Evans Blue does not enter the brain tissue. If the integrity of BBB is damaged or BBB's permeability increases, Evans Blue will infiltrate into the brain through the peripheral blood circulation to explore whether LPS induced systemic inflammation can cause damage to the BBB.
2, after immunofluorescence antibody labelled brain single cells, flow cytometry was used to analyze the infiltration of NK cells and other inflammatory cells to CNS, and the activation state of microglia in brain tissue was detected by immunofluorescence, in order to explore whether LPS could cause the inflammatory reaction of CNS.
3, using anti NK antibody (PK136) to scavenging peripheral NK cells and removing NK cells after LPS treatment for three times, LPS20 micrograms per day (hereinafter referred to as LPS+PK136 group), and detecting the body weight of mice at each LPS treatment. Flow cytometry was used to analyze the changes in immune cells in CNS, at the same time, the immunofluorescent detection of PBS group, LPS group and LPS+PK136 group brain tissue microglia cells Expression level and expression level of inflammatory factor mRNA in brain tissue.
4, in order to understand the cytological mechanism of NK cells in LPS induced nervous system inflammation, LPS was used to dynamically detect the infiltration of NK cells and other immune cells to CNS at different time points, to clear the peripheral NK cells in advance by anti NK antibody and to treat the LPS, and to detect the changes of neutrophils and mononuclear cells infiltrated by flow cytometry.
Result
1, after LPS treatment, the weight of mice decreased gradually, and then decreased to the lowest on the second, third day, then it showed an upward trend. Therefore, the experiment used LPS treatment for two days or three days of severe inflammation. After 4%Evans Blue treatment, the brain tissue of group LPS was stained, and the group PBS group had no color changes basically, indicating the integrity of BBB. This experiment proved that LPS induced systemic inflammation and also increased the permeability of BBB, suggesting that peripheral immune cells might infiltrate into CNS.
2, in the brain tissue flow analysis, a large number of leukocyte infiltration was found in CNS after LPS treatment, including NK cells, neutrophils and mononuclear cells significantly increased, suggesting that there was an inflammatory reaction in CNS at this time. Brain tissue immunofluorescence Iba1 staining found that the LPS group was more activated than the PBS group microglia. Furthermore, the mRNA detection of brain tissue found that LPS group was more than PB. The expression level of IL-1 in group S increased significantly. This experiment showed that intraperitoneal injection of LPS induced systemic inflammation and also caused inflammation of nervous system.
3, pre clearance of NK cells and intraperitoneal injection of LPS showed that the inflammatory cells infiltrated in the brain were significantly lower than those in the unscavenged NK group. The activation of microglia was significantly reduced, the body weight was significantly increased, and the expression of IL-1 decreased significantly. This experiment suggests that NK cells may play an inflammatory role in the inflammation of the nervous system induced by LPS.
4, in the LPS induced neuroinflammation model, the infiltration of NK cells in CNS was earlier than neutrophils. The neutrophils and mononuclear cells infiltrated in CNS were significantly reduced after the pre clearance of NK cells. This experiment showed that in the LPS induced neuroinflammatory model, the infiltrated NK cells may be induced by the attraction of peripheral neutrophils and mononuclear cells. Promote the inflammatory response of CNS.
conclusion
Intraperitoneal injection of LPS induces systemic systemic inflammation and can also induce inflammation in the nervous system, with the infiltration of large numbers of immune cells, the elimination of NK cells in the nervous system and the decrease of neutrophils in the brain, and the decrease in monocyte infiltration, indicating that NK cells are involved in the regulation of nervous system inflammation, which may be NK Cells enter the brain tissue to further promote the infiltration of peripheral neutrophils and monocytes into CNS.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

【参考文献】