本文選題：膿毒癥 + 腸屏障功能��； 參考：《第二軍醫(yī)大學(xué)》2014年博士論文

【摘要】：研究背景與目的 膿毒癥是機(jī)體對(duì)感染產(chǎn)生的一種全身炎癥反應(yīng)，如今仍是加強(qiáng)監(jiān)護(hù)病房引起患者死亡的主要原因之一。腸黏膜屏障功能障礙和通透性增加伴隨著膿毒癥的發(fā)生發(fā)展。腸黏膜屏障功能受損后引發(fā)的細(xì)菌移位可成為繼發(fā)膿毒癥的原因，而膿毒癥導(dǎo)致的腸組織血液供應(yīng)下降及炎癥因子風(fēng)暴可進(jìn)一步加重腸黏膜破壞，由此形成惡性循環(huán)。近來研究顯示免疫共抑制分子程序性死亡受體-1(programmed cell death-1, PD-1)/程序性死亡受體配體-1(programmed cell death ligand-1, PD-L1)信號(hào)通路與腸道免疫耐受及多種腸道炎性疾病相關(guān)；通過基因敲除小鼠或阻斷性抗體干預(yù)阻斷PD-1/PD-L1信號(hào)通路可以減輕膿毒癥導(dǎo)致的小鼠腸組織損傷。腸上皮細(xì)胞是腸黏膜物理屏障的重要組成部分，同時(shí)腸上皮細(xì)胞還可作為抗原遞呈細(xì)胞參與腸免疫屏障。PD-L1主要表達(dá)在抗原遞呈細(xì)胞，在非免疫細(xì)胞表面也有表達(dá)。研究顯示胃上皮及結(jié)腸上皮細(xì)胞均表達(dá)或可誘導(dǎo)表達(dá)PD-L1，，關(guān)于小腸上皮細(xì)胞是否表達(dá)PD-L1及其在膿毒癥導(dǎo)致的腸屏障功能障礙中的作用尚不明確。 本課題擬首先研究PD-L1分子在正常小鼠小腸上皮細(xì)胞的表達(dá)；其次觀察盲腸結(jié)扎穿孔(cecal ligation and puncture, CLP)對(duì)小鼠小腸上皮細(xì)胞PD-L1表達(dá)的影響。最后利用PD-L1基因敲除小鼠，觀察PD-L1敲除是否改善膿毒癥腸黏膜屏障功能障礙并探討可能的機(jī)制，并在體外用人腸上皮細(xì)胞系Caco-2細(xì)胞進(jìn)行驗(yàn)證。 實(shí)驗(yàn)對(duì)象及方法 1.正常八周齡雄性野生型C57BL/6小鼠4只，處死后分離小腸上皮細(xì)胞，應(yīng)用實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(ploymerase chain reaction, PCR)、Western blot分別檢測(cè)PD-L1mRNA和蛋白表達(dá)。 2.雄性野生型C57BL/6小鼠42只，隨機(jī)分為假手術(shù)組（Sham，n=18）和模型組（CLP, n=24）。分三個(gè)時(shí)間點(diǎn)（手術(shù)后6h、24h和48h）處死小鼠，分離小腸上皮細(xì)胞，應(yīng)用定量PCR、Western blot分別檢測(cè)PD-L1mRNA和蛋白表達(dá)。 3.將10只野生型C57BL/6小鼠及10只PD-L1基因敲除小鼠隨機(jī)分為WTSham組、WT CLP組、PD-L1-/-Sham組和PD-L1-/-CLP組，假手術(shù)組每組4只，模型組每組6只。術(shù)后24h檢測(cè)不同組小鼠小腸對(duì)4KD熒光素異硫氰酸酯-葡聚糖（fluorescein isothiocyanate dextran, FD4）的通透性反應(yīng)小腸黏膜屏障功能的變化。 4.將14只野生型C57BL/6小鼠及14只PD-L1基因敲除小鼠隨機(jī)分為WTSham組、WT CLP組、PD-L1-/-Sham組和PD-L1-/-CLP組，假手術(shù)組每組6只，模型組每組8只。術(shù)后24h收集小鼠回腸組織。利用ELISA方法檢測(cè)小腸組織勻漿內(nèi)TNF-α、IL-6、IL-10和MCP-1等細(xì)胞因子的表達(dá)。用Western bolt方法檢測(cè)小腸ZO-1、Occludin、Claudin-1等緊密連接蛋白的表達(dá)。 5.培養(yǎng)人結(jié)腸上皮細(xì)胞系Caco-2細(xì)胞，用定量PCR、Western blot和流式細(xì)胞術(shù)分別檢測(cè)Caco-2細(xì)胞PD-L1mRNA和蛋白表達(dá)，用定量PCR檢測(cè)重組人TNF-α或IFN-γ不同濃度和時(shí)間刺激后Caco-2細(xì)胞PD-L1mRNA表達(dá)，用流式細(xì)胞術(shù)檢測(cè)重組人TNF-α和IFN-γ聯(lián)合刺激后Caco-2細(xì)胞表面PD-L1蛋白表達(dá)。 6.利用Caco-2細(xì)胞建立腸上皮細(xì)胞屏障模型，分為對(duì)照組、TNF-α/IFN-γ預(yù)處理組、TNF-α/IFN-γ+抗PD-L1抗體處理組、TNF-α/IFN-γ+同型對(duì)照抗體處理組。測(cè)定不同組細(xì)胞單層接受處理72h后對(duì)FD-4的通透性；用免疫熒光染色觀察抗PD-L1抗體對(duì)Caco-2單層緊密連接蛋白ZO-1和Occludin的影響。 結(jié)果 1.小鼠小腸上皮細(xì)胞組成性表達(dá)PD-L1mRNA及蛋白。 2.膿毒癥小鼠小腸上皮細(xì)胞PD-L1mRNA表達(dá)在術(shù)后6h、24h和48h均顯著高于假手術(shù)組（p0.05），PD-L1蛋白表達(dá)在術(shù)后6h無變化，術(shù)后24h和48h與假手術(shù)組相比均顯著升高（p0.05）。 3.術(shù)后24h，與WT Sham組相比，野生型膿毒癥小鼠小腸對(duì)FD4的通透性顯著增加（p0.05），PD-L1基因敲除顯著降低了膿毒癥小鼠小腸對(duì)FD4的通透性（p0.05）。 4.術(shù)后24h，與WT Sham組相比，野生型膿毒癥小鼠回腸組織TNF-α、IL-6和MCP-1水平顯著升高（p0.05），ZO-1、Occludin、Claudin-1蛋白表達(dá)明顯下降（p0.05）。與WT CLP組相比，PD-L1基因敲除顯著降低了膿毒癥小鼠小腸組織內(nèi)TNF-α、IL-6和MCP-1等細(xì)胞因子水平（p0.05），PD-L1基因缺失可以預(yù)防膿毒癥導(dǎo)致的腸緊密連接蛋白ZO-1、Occludin、Claudin-1表達(dá)下降（p0.05）。 5.人腸上皮細(xì)胞系Caco-2細(xì)胞組成性表達(dá)PD-L1mRNA及蛋白。PD-L1mRNA及蛋白在重組人TNF-α和/或IFN-γ刺激后顯著增加（p0.05）。 6.抗PD-L1抗體預(yù)處理顯著減輕TNF-α和IFN-γ引起的Caco-2單層通透性增加（p0.05），同時(shí)能夠防止TNF-α和IFN-γ預(yù)處理引起的細(xì)胞間ZO-1和Occludin緊密連接蛋白結(jié)構(gòu)紊亂。 結(jié)論 小腸上皮細(xì)胞表達(dá)免疫共抑制分子PD-L1，膿毒癥顯著增加PD-L1在腸上皮細(xì)胞的表達(dá)。PD-L1基因敲除可明顯改善膿毒癥導(dǎo)致的小腸通透性增加，其機(jī)制可能為減少腸組織內(nèi)細(xì)胞因子水平，防止膿毒癥導(dǎo)致的緊密連接蛋白表達(dá)下降。研究提示腸上皮細(xì)胞表達(dá)PD-L1，參與膿毒癥導(dǎo)致的腸屏障功能紊亂。
[Abstract]:Background and purpose of study

Septicemia is one of the main causes of systemic inflammation caused by infection , and it is still one of the main causes of death in intensive care ward . Intestinal mucosal barrier dysfunction and increased permeability are associated with the development of sepsis . Bacterial translocation caused by intestinal mucosal barrier function can be the cause of secondary sepsis , and sepsis leads to a vicious circle . Recent studies have shown that the immune coinhibition molecule programmed cell death - 1 ( PD - 1 ) / programmed death receptor ligand - 1 ( programmed cell death ligand - 1 , PD - L1 ) signal pathway is associated with intestinal immune tolerance and various enteric diseases .
PD - 1 / PD - L1 signaling pathway can alleviate intestinal tissue injury in mice due to sepsis . Intestinal epithelial cells are an important part of the physical barrier of intestinal mucosa , and intestinal epithelial cells can also act as antigen - presenting cells to participate in intestinal immune barrier .

To study the expression of PD - L1 molecules in small intestinal epithelial cells of normal mice .
The effect of cecal ligation and puncture ( CLP ) on the expression of PD - L1 in small intestinal epithelial cells of mice was investigated .

Experimental object and method

1 . Four male wild - type C57BL / 6 mice were sacrificed and the small intestine epithelial cells were isolated . The expression of PD - L1 mRNA and protein was detected by real - time fluorescence quantitative polymerase chain reaction ( PCR ) and Western blot .

2 . Forty - two male wild - type C57BL / 6 mice were randomly divided into sham operation group ( Sham , n = 18 ) and model group ( CLP , n = 24 ) . Mice were sacrificed at three time points ( 6 h , 24 h and 48 h after surgery ) to separate small intestinal epithelial cells . Quantitative PCR and Western blot were used to detect PD - L1 mRNA and protein expression .

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本文編號(hào)：2087668