本文選題：神經(jīng)系統(tǒng)炎癥 + 小膠質(zhì)細胞��； 參考：《泰山醫(yī)學院》2014年碩士論文

【摘要】：目的 本實驗以腹腔注射脂多糖(Lipopolysaccharide, LPS)誘導的C57BL/6小鼠的神經(jīng)系統(tǒng)炎癥為模型，通過抗體清除外周及腦內(nèi)浸潤的自然殺傷細胞(Natural killer cells,NK)，檢測NK細胞在神經(jīng)炎癥中對小膠質(zhì)細胞活化和炎癥細胞浸潤的影響，以探討NK細胞在LPS誘導的神經(jīng)系統(tǒng)炎癥中的作用。 方法 1、動態(tài)觀察和監(jiān)測LPS處理不同時間點小鼠體重，繪制體重變化曲線以確定系統(tǒng)性炎癥的變化。于LPS或PBS處理兩次時同時尾靜脈注射4%Evans Blue200ul，次日用1%戊巴比妥鈉100ul麻醉小鼠，待其深入麻醉時用冰PBS心臟灌注，取腦觀察Evans Blue是否浸潤腦組織。正常情況下由于血腦屏障(Blood brain barrier，BBB)的存在，Evans Blue不能進入腦組織，若BBB的完整性遭到損傷或者BBB通透性增加時，Evans Blue會經(jīng)外周血液循環(huán)浸潤到腦內(nèi)。以此來探討LPS誘導的系統(tǒng)性炎癥能否夠引起B(yǎng)BB的破壞。 2、免疫熒光抗體標記腦單細胞后，流式分析NK細胞及其他炎癥細胞向CNS的浸潤，同時用免疫熒光檢測腦組織小膠質(zhì)細胞的活化狀態(tài)，以此來探討LPS能否引起CNS的炎癥反應。 3、用抗NK抗體(PK136)清除外周NK細胞，清除NK細胞后用LPS處理三次，LPS20微克/只/天(以下稱為LPS+PK136組)，并于每次LPS處理時檢測小鼠體重。用流式分析CNS中浸潤免疫細胞的變化，同時免疫熒光檢測PBS組、LPS組和LPS+PK136組腦組織小膠質(zhì)細胞Iba1的表達情況和腦組織炎性因子mRNA的表達水平。 4、為了解NK細胞在LPS誘導的神經(jīng)系統(tǒng)炎癥中的細胞學機制，動態(tài)檢測LPS處理不同時間點NK細胞及其他免疫細胞向CNS的浸潤，用抗NK抗體預先清除外周NK細胞后進而LPS處理，流式檢測浸潤的中性粒細胞和單核細胞的變化。 結(jié)果 1、LPS處理后小鼠體重逐步減輕，于第二、三天降至最低，隨后又呈上升趨勢，故本實驗采用LPS處理二天或三天炎癥較重時進行檢測。經(jīng)4%Evans Blue處理后發(fā)現(xiàn)LPS組的腦組織被染料著色，，而PBS組腦組織基本上沒有顏色變化，說明BBB的完整性遭到破壞。此實驗證明LPS誘導系統(tǒng)性炎癥的同時也引起了BBB通透性的增加，提示外周的免疫細胞可能會浸潤到CNS中。 2、在腦組織流式分析中發(fā)現(xiàn)LPS處理后CNS中有大量白細胞浸潤，包括NK細胞、中性粒細胞和單核細胞顯著增加，提示此時CNS內(nèi)發(fā)生了炎癥反應。腦組織免疫熒光Iba1染色發(fā)現(xiàn)LPS組較PBS組小膠質(zhì)細胞明顯活化。此外，腦組織mRNA檢測發(fā)現(xiàn)LPS組比PBS組IL-1表達水平顯著性增加。此實驗說明腹腔注射LPS誘導系統(tǒng)性炎癥的同時也引起了神經(jīng)系統(tǒng)的炎癥。 3、預先清除NK細胞后進而LPS腹腔注射，發(fā)現(xiàn)腦內(nèi)浸潤的炎癥細胞較未清除NK組顯著性降低，小膠質(zhì)細胞活化明顯減輕，體重顯著性增加，IL-1表達也顯著性降低。此實驗提示NK細胞在LPS誘導的神經(jīng)系統(tǒng)炎癥中可能起促炎作用。 4、在LPS誘導的神經(jīng)炎癥模型中，CNS中NK細胞的浸潤早于中性粒細胞。預先清除NK細胞后CNS中浸潤的中性粒細胞和單核細胞顯著性降低。此實驗說明在LPS誘導的神經(jīng)炎性模型中，浸潤的NK細胞可能通過吸引外周中性粒細胞和單核細胞進而促進CNS的炎癥反應。 結(jié)論 腹腔注射LPS引起全身系統(tǒng)性炎癥的同時也可以誘導神經(jīng)系統(tǒng)發(fā)生炎癥，伴隨大量免疫細胞的浸潤，清除其中的NK細胞導致神經(jīng)系統(tǒng)的炎癥減輕和腦內(nèi)中性粒細胞、單核細胞浸潤減少，說明NK細胞參與調(diào)控神經(jīng)系統(tǒng)炎癥，這種調(diào)控可能是NK細胞進入腦組織后進一步促進外周中性粒細胞和單核細胞向CNS浸潤而實現(xiàn)。
[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 .
【學位授予單位】：泰山醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741

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相關(guān)期刊論文 前2條

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2 薛e

本文編號：2097263