發(fā)布時(shí)間：2018-02-20 20:52

  本文關(guān)鍵詞： 高遷移率族蛋白B1 活化T細(xì)胞的核因子 白介素-2 免疫反應(yīng) 信號(hào)轉(zhuǎn)導(dǎo) 膿毒癥　出處：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 高遷移率族蛋白B1(high mobility group box-1 protein，HMGB1)是一大類高度保守的非組蛋白DNA結(jié)合蛋白，不僅廣泛存在于真核細(xì)胞的細(xì)胞核內(nèi)，對(duì)DNA復(fù)制、轉(zhuǎn)錄、重組和修復(fù)具有重要作用，而且可作為一種新的“晚期”炎癥介質(zhì)由單核/巨噬細(xì)胞分泌到細(xì)胞外參與炎癥反應(yīng)。新近研究表明，HMGB1不僅與機(jī)體的炎癥反應(yīng)密切相關(guān)，還可以作為一種免疫調(diào)節(jié)因子引起淋巴細(xì)胞激活并釋放白介素(IL)-2，誘導(dǎo)樹突狀細(xì)胞活化并分泌IL-12。而且，我們前期實(shí)驗(yàn)證實(shí)，在小鼠淋巴細(xì)胞內(nèi)加入重組HMGB1可引起淋巴細(xì)胞大量凋亡，并可影響活化T細(xì)胞核因子(nuclear factor of activated T cells，NFAT)的入核啟動(dòng)基因轉(zhuǎn)錄，IL-2水平亦受到影響。但是，目前HMGB1以何種機(jī)制影響T淋巴細(xì)胞合成釋放IL-2還不明確，其中的確切信號(hào)轉(zhuǎn)導(dǎo)途徑尚缺乏研究。 已證實(shí)，NFAT家族是淋巴細(xì)胞內(nèi)免疫應(yīng)答的主要信號(hào)通路。NFAT受T淋巴細(xì)胞受體(TCR)信號(hào)活化后，轉(zhuǎn)位入核內(nèi)調(diào)控重要基因轉(zhuǎn)錄表達(dá)。NFATs家族已發(fā)現(xiàn)有5個(gè)成員，包括NFAT1(NFATp，NFATc2)、NFAT2(NFATc1，NFATc)、NFAT3(NFATc4)、NFAT4(NFATc3，NFATx)及NFAT5(TonEBP，tonicity element binding protein，緊張片斷結(jié)合蛋白)。NFAT1-4是由鈣調(diào)節(jié)的，與細(xì)胞的免疫調(diào)節(jié)反應(yīng)關(guān)系密切；NFAT5相對(duì)原始，是NFATs家族中唯一可在果蠅基因組中找到的。研究表明，成熟的T淋巴細(xì)胞主要表達(dá)NFAT1、NFAT2，是影響IL-2合成釋放的關(guān)鍵轉(zhuǎn)錄因子。因此，HMGB1有可能通過與NFAT1或NFAT2蛋白的相互作用進(jìn)一步影響IL-2的合成釋放。本實(shí)驗(yàn)擬構(gòu)建HMGB1與NFAT2的真核表達(dá)質(zhì)粒，驗(yàn)證二者是否存在協(xié)同效應(yīng)?并通過促進(jìn)IL-2報(bào)告基因的表達(dá)水平分析，進(jìn)一步檢測(cè)二者之間的相互結(jié)合作用；此外，初步探討二者之間的相互作用位點(diǎn)，反過來再次驗(yàn)證其確切相互作用。該研究旨在從信號(hào)轉(zhuǎn)導(dǎo)水平初步了解HMGB1影響IL-2表達(dá)釋放的分子機(jī)制，豐富膿毒癥時(shí)機(jī)體免疫調(diào)節(jié)異常改變的認(rèn)識(shí)，為膿毒癥的有效干預(yù)和防治提供新思路。 本實(shí)驗(yàn)采用PCR、co-IP、Western-blot、GST-pull down及sRNAi的方法首先證實(shí)了HMGB1與NFAT2之間的確存在相互協(xié)同作用，可顯著提高IL-2報(bào)告基因的表達(dá)活性。然后在此基礎(chǔ)之上，觀察了二者在體外和細(xì)胞內(nèi)的相互直接結(jié)合作用，并初步明確了HMGB1與NFAT2結(jié)合的具體位點(diǎn)。為將來設(shè)計(jì)位點(diǎn)干擾的免疫調(diào)節(jié)藥物奠定了良好基礎(chǔ)。 主要結(jié)果和結(jié)論如下： 1．HMGB1可與NFAT2協(xié)同促進(jìn)IL-2表達(dá)，二者協(xié)同促進(jìn)報(bào)告基因活性上升的倍數(shù)遠(yuǎn)大于二者單獨(dú)作用時(shí)倍數(shù)之和。 2．IL-2報(bào)告基因活性得到充分提高必須依賴于HMGB1和NFAT2共同作用，，二者缺一不可。逐步增加HMGB1轉(zhuǎn)染量，可劑量依賴性增強(qiáng)報(bào)告基因的活性。應(yīng)用sRNAi技術(shù)抑制二者任一方都可使IL-2報(bào)告基因活性下降。 3．HMGB1和NFAT2之間存在直接的相互作用，應(yīng)用GST-pull down實(shí)驗(yàn)及co-IP實(shí)驗(yàn)證實(shí)二者在細(xì)胞外和細(xì)胞內(nèi)都可以直接相互結(jié)合。 4．初步明確HMGB1是通過B box與NFAT2相互結(jié)合，而A box不能與NFAT2在體外結(jié)合。
[Abstract]:High mobility group box - 1 protein is a highly conserved non - histone DNA binding protein , which is not only widely present in the nucleus of eukaryotic cell , but also plays an important role in DNA replication , transcription , recombination and repair . The NFAT family has been confirmed to be the main signaling pathway for the immune response in lymphocytes . NFAT has been activated by T lymphocyte receptor ( TCR ) signal . The NFAT family has been found to have 5 members , including NFAT1 ( NFATp , NFATc2 ) , NFAT2 ( NFATc1 , NFATc ) , NFAT3 ( NFATc4 ) , NFAT4 ( NFATc3 , NFATx ) and NFAT5 ( TonEBP , NFATx ) and NFAT5 ( TonEBP , NFATx ) and NFAT5 ( TonEBP , NFATx ) and NFAT5 ( TonEBP , NFATx ) . It was found that NFAT5 was the only one of NFAT5 and NFAT2 , which was the only key transcription factor affecting the release of IL - 2 . Using PCR , co - IP , Western - blot , GST - pull down and sRNAi methods , it was confirmed that there was a synergistic effect between the two groups , which could significantly improve the expression of IL - 2 reporter gene . The main results and conclusions are as follows : 1 . The expression of IL - 2 can be promoted in conjunction with NFAT2 . Both of them can promote the expression of IL - 2 in combination with NFAT2 . 2 . The activity of IL - 2 reporter gene must be increased and the activity of IL - 2 reporter gene can be enhanced by increasing the transfection dose and increasing the activity of the reporter gene . The activity of IL - 2 reporter gene can be decreased by using sRNAi technology . 3 . There was a direct interaction between NF - AT2 , and the GST - pull down experiment and co - IP experiment were used to confirm that both were able to bind directly to each other both in vitro and in the cell . 4 . It is preliminarily clear that the combination of B box and NFAT2 , while A box cannot be combined with NFAT2 in vitro .

【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 徐姍;姚詠明;董寧;劉峰;盛志勇;;高遷移率族蛋白B1對(duì)大鼠脾臟樹突狀細(xì)胞表面共刺激分子表達(dá)的影響[J];中華創(chuàng)傷雜志;2006年08期

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