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]:Purpose

In this study , the effects of NK cells on activation of microglial cells and infiltration of inflammatory cells were investigated in C57BL / 6 mice induced by intraperitoneal injection of lipopolysaccharide ( LPS ) , and the role of NK cells in LPS - induced nervous system inflammation was investigated .

method

1 . To observe and monitor the body weight of mice at different time points in LPS or PBS to determine the changes of systemic inflammation . After treated with LPS or PBS twice , 4 % Evans Blue 200ul was injected intravenously . After the treatment , Evans Blue could not enter the brain tissue . When the integrity of BBB was damaged or the BBB permeability increased , Evans Blue could infiltrate into the brain via peripheral blood circulation .

2 . After labeling the brain cells with immunofluorescence antibody , the infiltration of NK cells and other inflammatory cells to the CNS was analyzed by flow cytometry , and the activation status of microglial cells in brain tissue was detected by immunofluorescence , which was used to investigate whether LPS could cause CNS inflammatory response .

3 . NK cells were removed by anti - NK antibody ( PK136 ) , NK cells were removed and treated with LPS three times , LPS20 ug / day ( hereinafter referred to as LPS + PK136 group ) , and the weight of mice was detected at each LPS treatment . The expression of Iba1 in brain tissue of PBS group , LPS group and LPS + PK136 group were detected by flow analysis , and the expression level of inflammatory factor mRNA in brain tissue was detected .

4 . To understand the cellular mechanism of NK cells in LPS - induced nervous system inflammation , the infiltration of NK cells and other immune cells to the CNS at different time points was dynamically detected by LPS .

Results

1 . After LPS treatment , the body weight of the mice was gradually decreased , and then decreased to the lowest in the second and three days , and then increased . After 4 % Evans Blue treatment , it was found that the brain tissue of LPS group was stained with dye , while the brain tissue of PBS group had no color change , which indicated that the integrity of BBB was destroyed . This experiment proves that LPS - induced systemic inflammation also leads to the increase of BBB permeability , suggesting that peripheral immune cells may infiltrate into the CNS .

2 . In the brain tissue flow analysis , there was a significant increase of leukocyte infiltration in the CNS after LPS treatment , including NK cells , neutrophils and monocytes , suggesting a significant increase in the expression of IL - 1 in the LPS group than in PBS group .

3 . After the NK cells were removed in advance , LPS was injected intraperitoneally . It was found that the inflammatory cells infiltrated in the brain were significantly lower than those in the uncleared NK group , the activation of microglial cells was significantly decreased , the body weight was significantly increased , and the expression of IL - 1 was significantly decreased . This experiment suggested that NK cells might play pro - inflammatory effects in LPS - induced nervous system inflammation .

4 . In the LPS - induced neuroinflammatory model , the infiltration of NK cells in the CNS was earlier than that of neutrophils . There was a significant decrease in the infiltration of neutrophils and monocytes in the CNS after pre - clearance of NK cells . This experiment shows that the infiltrated NK cells may contribute to the inflammatory response of the CNS by attracting peripheral neutrophils and monocytes in LPS - induced neuroinflammatory models .

Conclusion

Intraperitoneal injection of LPS can induce systemic inflammation and induce inflammation in the nervous system , accompanied by infiltration of a large number of immune cells . NK cells can be removed to reduce inflammation of the nervous system and the infiltration of neutrophils and monocytes in the brain . NK cells may be involved in the regulation of nervous system inflammation , which may be achieved by further promoting the infiltration of peripheral neutrophils and monocytes into the CNS after the NK cells enter the brain tissue .
【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】