本文選題：Tim3 + 巨噬細(xì)胞　； 參考：《河南大學(xué)》2011年碩士論文

【摘要】：背景:T細(xì)胞免疫球蛋白及粘蛋白家族-3(Tim3)作為Tim家族的成員,有281個(gè)氨基酸組成,最先被發(fā)現(xiàn)特異的表達(dá)在CD4+ Th1細(xì)胞上,而不在Th2細(xì)胞上表達(dá)。這個(gè)發(fā)現(xiàn)促使人們?nèi)パ芯縏im3是如何調(diào)節(jié)Th1型細(xì)胞反應(yīng),又是如何在自身免疫性疾病中發(fā)揮作用? Tim-3分子通過(guò)負(fù)性調(diào)節(jié)Th1細(xì)胞的功能廣泛參與了特異性免疫應(yīng)答的調(diào)節(jié)。近期研究發(fā)現(xiàn),Tim-3在DC、單核細(xì)胞、巨噬細(xì)胞等天然免疫細(xì)胞表面也有表達(dá),通過(guò)激活炎癥細(xì)胞發(fā)揮促炎癥作用。 目的:探討Tim-3對(duì)巨噬細(xì)胞功能的調(diào)節(jié)作用及其機(jī)制。 方法:(1)首先將含Tim-3SiRNA及NC對(duì)照序列的載體經(jīng)脂質(zhì)體分別轉(zhuǎn)染細(xì)胞系RAW264.7,以G418加壓篩選,通過(guò)RNA干擾降低Tim3在巨噬細(xì)胞表面的表達(dá),構(gòu)建了一種穩(wěn)定低表達(dá)Tim3的RAW264.7巨噬細(xì)胞系。以野生型及低表達(dá)Tim3的巨噬細(xì)胞為研究對(duì)象,探討Tim-3對(duì)巨噬細(xì)胞活性及表型的影響。(2)體外阻斷或激活Tim3通路,ELISA法檢測(cè)巨噬細(xì)胞等天然免疫細(xì)胞分泌炎癥因子TNF-α、IL-6的水平;通過(guò)Western blot方法檢測(cè)炎癥反應(yīng)信號(hào)通路中關(guān)鍵分子AKT、PAKT、NF-kB的變化;通過(guò)ELISA法、Western blot檢測(cè)Tim-3與模式識(shí)別受體TLR4的相互作用。雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)NF-kB的活性。通過(guò)以上方法檢測(cè)Tim3參與巨噬細(xì)胞炎癥反應(yīng)的機(jī)制。(3)建立敗血癥動(dòng)物模型,觀察Tim3在敗血癥早期和晚期的不同作用。(4)以敗血癥小鼠模型為基礎(chǔ),Tim-3體內(nèi)阻斷實(shí)驗(yàn)檢測(cè)Th1、Th17細(xì)胞活性,研究Tim-3對(duì)Th1,Th17的分化的影響以及對(duì)小鼠腹腔巨噬細(xì)胞表面共刺激分子CD80、CD86的表達(dá)的影響。同時(shí)體外實(shí)驗(yàn)檢測(cè)Tim3對(duì)巨噬細(xì)胞表面共刺激分子CD86/CD80表達(dá)比例的影響以及對(duì)Th1,Th17的分化的影響。從體內(nèi)實(shí)驗(yàn)和體外實(shí)驗(yàn)兩方面探討Tim3對(duì)巨噬細(xì)胞抗原呈遞作用及對(duì)Th1,Th17細(xì)胞極化的影響。 結(jié)果:(1)成功構(gòu)建了重組載體Tim3-siRNA,建立了穩(wěn)定低表達(dá)Tim3的巨噬細(xì)胞系。(2)為了探討Tim-3在天然免疫中的調(diào)節(jié)作用,我們觀察了Tim-3對(duì)巨噬細(xì)胞活性的影響,結(jié)果顯示Tim-3刺激巨噬細(xì)胞分泌炎癥因子TNF-α、IL-6。同時(shí)ELISA結(jié)果顯示Tim3通路和TLR4通路在刺激巨噬細(xì)胞釋放炎性因子TNF-α方面存在相互拮抗效應(yīng)。Western blot結(jié)果也顯示Tim3信號(hào)通路和TLR4信號(hào)通路的相互拮抗關(guān)系涉及到對(duì)一些重要的信號(hào)分子的影響,如AKT活性過(guò)強(qiáng)抑制了NF-kB活性,從而抑制了LPS介導(dǎo)的TNF-α的分泌。(3)體內(nèi)實(shí)驗(yàn)結(jié)果顯示,以敗血癥小鼠模型為基礎(chǔ),Tim-3在敗血癥早期表達(dá)升高,發(fā)揮抗炎作用,而在敗血癥后期表達(dá)失調(diào)。(4)體內(nèi)體外實(shí)驗(yàn)結(jié)果顯示,Tim-3在提高CD86,降低CD80,提高CD86/CD80的比例中發(fā)揮重要作用。同時(shí)Tim-3可以促進(jìn)Th1及Th17細(xì)胞的分化,表現(xiàn)為Th1型細(xì)胞因子IFN-γ,以及Th17型細(xì)胞因子IL-17表達(dá)的升高。 小結(jié):(1)Tim3通過(guò)促使巨噬細(xì)胞分泌炎癥因子TNF-α、IL-6參與了天然免疫反應(yīng)調(diào)節(jié)。(2)Tim3通路和TLR4信號(hào)通路在介導(dǎo)炎癥反應(yīng)方面存在相互拮抗效應(yīng)。(3)Tim3參與了巨噬細(xì)胞表面CD80,CD86共刺激分子的調(diào)節(jié),Tim-3通過(guò)提高CD86/CD80的比例促進(jìn)Th1及Th17細(xì)胞的分化。(4)本實(shí)驗(yàn)為認(rèn)識(shí)Tim-3在天然免疫和獲得性免疫中的作用提供了新的機(jī)制,為Tim3在巨噬細(xì)胞介導(dǎo)的免疫反應(yīng)中的調(diào)節(jié)作用提供了新的理論依據(jù)。
[Abstract]:Background : T - cell immunoglobulin and mucin family - 3 ( Tim3 ) , a member of the Tim family , have 281 amino acids , which were first found to be specifically expressed on CD4 + Th1 cells and not on Th2 cells . This finding has prompted people to investigate how Tim3 regulates Th1 - type cell responses and how to play a role in autoimmune diseases ? Tim - 3 has been extensively involved in the regulation of specific immune responses by negatively regulating the function of Th1 cells . Recent studies have shown that Tim - 3 also has an expression on the surface of natural immune cells such as DC , monocytes , macrophages , etc . , which exerts a pro - inflammatory effect by activating inflammatory cells .


Objective : To investigate the effect of Tim - 3 on macrophage function and its mechanism .


Methods : ( 1 ) The effects of Tim - 3 on the activity and phenotype of macrophages were investigated by means of Western blot . The effects of Tim - 3 on the activity and phenotype of macrophages were investigated by means of Western blot .


緇撴灉:(1)鎴愬姛鏋勫緩浜?jiǎn)閲嵕l勮澆浣揟im3-siRNA,寤虹珛浜?jiǎn)绋冲畾浣庤〃杈綯im3鐨勫法鍣粏鑳?yōu)绯?(2)涓轟簡(jiǎn)鎺㈣Tim-3鍦ㄥぉ鐒跺厤鐤腑鐨勮皟鑺備綔鐢，

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