發(fā)布時(shí)間：2018-05-26 03:46

  本文選題：T細(xì)胞活化連接蛋白（LAT） + 細(xì)胞功能�。� 參考：《上海交通大學(xué)》2012年博士論文

【摘要】：T細(xì)胞活化連接蛋白（LAT）是TCR與MHC-抗原復(fù)合物結(jié)合后，在T細(xì)胞抗原受體（TCR）傳遞活化信號(hào)早期階段發(fā)揮關(guān)鍵作用的跨膜連接蛋白，是ZAP-70等Src家族蛋白酪氨酸激酶的重要作用底物。LAT分子胞內(nèi)段酪氨酸殘基被ZAP-70磷酸化后，可以招募諸多胞內(nèi)游離分子如Grb2, PLC-γ1，Gads等，一方面被招募的PLC-1被活化后通過水解PIP2產(chǎn)生重要信使分子IP3和DAG，啟動(dòng)鈣離子內(nèi)流和激活Ras-MAPK等途徑；同時(shí)被招募的Gads可以進(jìn)一步和SLP-76相結(jié)合，參與細(xì)胞骨架重塑和整合素活性升高，LAT還可以通過結(jié)合Grb2-SOS分子并招募其到細(xì)胞膜從而實(shí)現(xiàn)對(duì)Ras-MAPK途徑的活化。上述這些直接或間接被LAT招募后所形成的“LAT信號(hào)復(fù)合體”（LAT Signalsome）實(shí)現(xiàn)了來自于抗原的胞外信號(hào)經(jīng)TCR識(shí)別后轉(zhuǎn)化成胞內(nèi)活化信號(hào)，最終實(shí)現(xiàn)T細(xì)胞的發(fā)育、成熟、增殖、活化和分化。 來自于LAT基因敲除小鼠實(shí)驗(yàn)?zāi)Ｐ偷难芯拷Y(jié)果證明LAT分子在T細(xì)胞發(fā)育中的關(guān)鍵作用， LAT分子缺失的T細(xì)胞系的研究顯示LAT分子在T細(xì)胞活化中的重要作用，但是來自于LATY136F突變小鼠和條件性剔除外周T細(xì)胞中l(wèi)at小鼠的研究結(jié)果提示在生理情況下LAT分子在成熟T細(xì)胞中表現(xiàn)出和胸腺發(fā)育過程中不同的生物學(xué)功能，，主要表現(xiàn)為TCR下游信號(hào)的衰減和淋巴細(xì)胞的異常增殖和分化。為此，我們推測(cè)LAT分子在外周成熟T細(xì)胞中還具有不為所知的新的調(diào)節(jié)T細(xì)胞功能作用模式，并對(duì)此進(jìn)行了本項(xiàng)目的研究。 在本研究中，我們通過Amaxa電轉(zhuǎn)體系將LAT特異性小片段干擾RNA經(jīng)體外轉(zhuǎn)染人外周成熟T細(xì)胞，建立瞬時(shí)高效干擾LAT分子表達(dá)系統(tǒng)，獲得LAT分子缺失原代T細(xì)胞和T細(xì)胞系，繼而開展功能試驗(yàn)探討LAT分子對(duì)T細(xì)胞功能的調(diào)節(jié)作用。在成功干擾LAT蛋白的表達(dá)之后，我們首先檢測(cè)了T細(xì)胞增殖以及細(xì)胞因子分泌等表型，結(jié)果顯示LAT分子缺失的T細(xì)胞在TCR/CD28雙信號(hào)刺激下，T細(xì)胞的增殖能力雖然沒有顯著改變，但是其IL-2的分泌較對(duì)照組有顯著的升高。同時(shí)，研究結(jié)果顯示IFN-的mRNA水平和分泌量則較對(duì)照組有所降低，IL-4的mRNA水平升高，提示LAT缺失的T細(xì)胞經(jīng)TCR/CD28活化后具有向Th2細(xì)胞分化的趨勢(shì)。 為了進(jìn)一步探討LAT分子缺失后導(dǎo)致的T細(xì)胞細(xì)胞應(yīng)答格局改變的分子機(jī)制，我們進(jìn)一步分析TCR介導(dǎo)的信號(hào)傳導(dǎo)通路中相關(guān)重要信號(hào)分子的磷酸化表達(dá)譜的改變。結(jié)果顯示，在LAT分子缺失的原代T細(xì)胞經(jīng)TCR/CD28激活后，位于LAT上游的Lck分子的磷酸化沒有改變，但位于LAT分子下游的PLC-γ1的磷酸化水平明顯下降，并導(dǎo)致鈣離子內(nèi)流的缺失；同時(shí)Erk1/2活化程度顯著降低。相反，研究結(jié)果發(fā)現(xiàn)LAT敲除后的T細(xì)胞經(jīng)TCR/CD28活化過程中，胞內(nèi)3，4，5三磷酸磷脂酰肌醇(PIP3)出現(xiàn)大量的聚集，該分子是CD28分子參與PI3K/AKT信號(hào)通路重要的活化信使分子，從而導(dǎo)致PI3K/AKT信號(hào)通路的增強(qiáng)和轉(zhuǎn)錄因子NF-B的持續(xù)磷酸化。 綜合上述研究結(jié)果，我們認(rèn)為L(zhǎng)AT分子在外周成熟T細(xì)胞還可能發(fā)揮調(diào)節(jié)其他信號(hào)通路如CD28信號(hào)通路的作用，其中PIP2可能是參與TCR信號(hào)途徑與CD28信號(hào)途徑轉(zhuǎn)換的關(guān)鍵鏈接分子，在正常表達(dá)LAT分子的T細(xì)胞中，來自于TCR/CD28的信號(hào)可以促進(jìn)PIP2在PLC-1的作用下水解為IP3和DAG，傳遞正常的TCR活化信號(hào)；而在LAT分子缺失的情況下，胞內(nèi)PIP2則向PIP3轉(zhuǎn)換，從而增強(qiáng)CD28介導(dǎo)的PI3K/AKT信號(hào)通路，以旁路激活的方式介導(dǎo)T細(xì)胞功能的改變，上述研究結(jié)果首次證實(shí)了LAT分子在成熟T細(xì)胞中的新的調(diào)節(jié)模式，為其所參與的TCR信號(hào)轉(zhuǎn)導(dǎo)通路參與調(diào)節(jié)其他信號(hào)通路提供了直接證據(jù)。
[Abstract]:T - cell activation connexin ( LAT ) is a transmembrane connexin which plays a key role in the early stage of T cell antigen receptor ( TCR ) transfer activation signal after binding of TCR to MHC - antigen complex . After phosphorylation of ZAP - 70 , intracellular free molecules such as Grb2 , PLC - 緯1 , Gads , etc . can be recruited . On the one hand , PLC - 1 is activated to produce important messenger molecule IP3 and DAG , activate calcium ion influx and activate Ras - MAPK .
At the same time , the recruited Gads may be further combined with SLP - 76 to participate in cytoskeletal remodeling and integrin activity , and LAT may also activate the Ras - MAPK pathway by binding to the Grb2 - SOS molecules and recruiting them to the cell membrane . These direct or indirect LAT Signalsome , which are directly or indirectly recruited , enable the conversion of extracellular signals from the antigen to intracellular activation signals after TCR recognition , ultimately to the development , maturation , proliferation , activation and differentiation of T cells .

The results suggest that LAT molecules play an important role in T cell activation , but the research results from lat mice of LAT molecule deletion in mature T cells show that LAT molecules exhibit different biological functions in mature T cells , which are mainly characterized by the attenuation of downstream signals of TCR and abnormal proliferation and differentiation of lymphocytes .

In this study , we investigated the effect of LAT molecule on T cell function in vitro . After successfully interfering with the expression of LAT protein , we first examined T cell proliferation and cytokine secretion . The results showed that the mRNA level and secretion of the LAT molecule were lower than those of the control group . The results showed that the mRNA level and secretion of the LAT molecule were lower than those of the control group .

In order to further study the molecular mechanism of T cell response pattern after LAT molecule deletion , we further analyzed the change of phosphorylation expression profile of relevant important signal molecules in TCR - mediated signal transduction pathways . The results showed that the phosphorylation of Lck molecules located upstream of LAT was not changed after activation of TCR / CD28 in primary T cells with LAT molecule deletion , but the phosphorylation level of PLC - 緯1 downstream of LAT molecule decreased obviously , and resulted in the deletion of calcium ion influx ;
At the same time , the activation degree of the Erk1 / 2 decreased significantly . On the contrary , the results showed that during the activation of TCR / CD28 , the intracellular 3 , 4 , 5 - triphosphate ( PIP3 ) produced a large amount of aggregation , which was the important activation messenger molecule of the CD28 molecule in the pathway of the 3 - 3 , 4 , 5 - triphosphate ( PIP3 ) , thus leading to the enhancement of the pathway and the continuous phosphorylation of NF - B .

Based on the above results , we think that LAT molecule may play a role in regulating other signaling pathways such as CD28 signaling pathway in peripheral mature T cells , where PIP2 may be a key link molecule involved in TCR signaling pathway and CD28 signaling pathway . In T cells expressing LAT molecules , the signal from TCR / CD28 can promote PIP2 to hydrolyze to IP3 and DAG under the action of PLC - 1 , delivering normal TCR activation signal ;
However , in the absence of LAT molecules , the intracellular PIP2 is converted to PIP3 , thereby enhancing the CD28 - mediated T cell function . The results showed that the new regulatory model of the LAT molecule in mature T cells was confirmed for the first time , and direct evidence was provided for the involvement of the TCR signal transduction pathways involved in the regulation of other signal pathways .
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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相關(guān)期刊論文 前1條

1 朱香清;陳剛;;PKB/Akt-NF-κB通路與糖尿病血管內(nèi)皮細(xì)胞功能的調(diào)節(jié)[J];國際內(nèi)科學(xué)雜志;2007年10期

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