本文選題：神經(jīng)系統(tǒng)炎癥 + 小膠質(zhì)細(xì)胞　； 參考：《泰山醫(yī)學(xué)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(dǎo)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(xì)胞(Natural killer cells,NK)，檢測NK細(xì)胞在神經(jīng)炎癥中對小膠質(zhì)細(xì)胞活化和炎癥細(xì)胞浸潤的影響，以探討NK細(xì)胞在LPS誘導(dǎo)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(jìn)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(dǎo)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(biāo)記腦單細(xì)胞后，流式分析NK細(xì)胞及其他炎癥細(xì)胞向CNS的浸潤，同時用免疫熒光檢測腦組織小膠質(zhì)細(xì)胞的活化狀態(tài)，以此來探討LPS能否引起CNS的炎癥反應(yīng)。 3、用抗NK抗體(PK136)清除外周NK細(xì)胞，清除NK細(xì)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(xì)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(xì)胞Iba1的表達(dá)情況和腦組織炎性因子mRNA的表達(dá)水平。 4、為了解NK細(xì)胞在LPS誘導(dǎo)的神經(jīng)系統(tǒng)炎癥中的細(xì)胞學(xué)機(jī)制，動態(tài)檢測LPS處理不同時間點NK細(xì)胞及其他免疫細(xì)胞向CNS的浸潤，用抗NK抗體預(yù)先清除外周NK細(xì)胞后進(jìn)而LPS處理，流式檢測浸潤的中性粒細(xì)胞和單核細(xì)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(jìn)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(dǎo)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(xì)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(xì)胞浸潤，包括NK細(xì)胞、中性粒細(xì)胞和單核細(xì)胞顯著增加，提示此時CNS內(nèi)發(fā)生了炎癥反應(yīng)。腦組織免疫熒光Iba1染色發(fā)現(xiàn)LPS組較PBS組小膠質(zhì)細(xì)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(dá)水平顯著性增加。此實驗說明腹腔注射LPS誘導(dǎo)系統(tǒng)性炎癥的同時也引起了神經(jīng)系統(tǒng)的炎癥。 3、預(yù)先清除NK細(xì)胞后進(jìn)而LPS腹腔注射，發(fā)現(xiàn)腦內(nèi)浸潤的炎癥細(xì)胞較未清除NK組顯著性降低，小膠質(zhì)細(xì)胞活化明顯減輕，體重顯著性增加，IL-1表達(dá)也顯著性降低。此實驗提示NK細(xì)胞在LPS誘導(dǎo)的神經(jīng)系統(tǒng)炎癥中可能起促炎作用。 4、在LPS誘導(dǎo)的神經(jīng)炎癥模型中，，CNS中NK細(xì)胞的浸潤早于中性粒細(xì)胞。預(yù)先清除NK細(xì)胞后CNS中浸潤的中性粒細(xì)胞和單核細(xì)胞顯著性降低。此實驗說明在LPS誘導(dǎo)的神經(jīng)炎性模型中，浸潤的NK細(xì)胞可能通過吸引外周中性粒細(xì)胞和單核細(xì)胞進(jìn)而促進(jìn)CNS的炎癥反應(yīng)。 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(dǎo)神經(jīng)系統(tǒng)發(fā)生炎癥，伴隨大量免疫細(xì)胞的浸潤，清除其中的NK細(xì)胞導(dǎo)致神經(jīng)系統(tǒng)的炎癥減輕和腦內(nèi)中性粒細(xì)胞、單核細(xì)胞浸潤減少，說明NK細(xì)胞參與調(diào)控神經(jīng)系統(tǒng)炎癥，這種調(diào)控可能是NK細(xì)胞進(jìn)入腦組織后進(jìn)一步促進(jìn)外周中性粒細(xì)胞和單核細(xì)胞向CNS浸潤而實現(xiàn)。
[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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 吳原;王學(xué)峰;;單核細(xì)胞趨化蛋白-1在CNS疾病中的表達(dá)[J];臨床神經(jīng)電生理學(xué)雜志;2006年05期

2 薛e

本文編號：2097262