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

【摘要】：T細胞活化連接蛋白（LAT）是TCR與MHC-抗原復合物結合后，在T細胞抗原受體（TCR）傳遞活化信號早期階段發(fā)揮關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分子胞內段酪氨酸殘基被ZAP-70磷酸化后，可以招募諸多胞內游離分子如Grb2, PLC-γ1，Gads等，一方面被招募的PLC-1被活化后通過水解PIP2產生重要信使分子IP3和DAG，啟動鈣離子內流和激活Ras-MAPK等途徑；同時被招募的Gads可以進一步和SLP-76相結合，參與細胞骨架重塑和整合素活性升高，LAT還可以通過結合Grb2-SOS分子并招募其到細胞膜從而實現對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信號復合體”（LAT Signalsome）實現了來自于抗原的胞外信號經TCR識別后轉化成胞內活化信號，最終實現T細胞的發(fā)育、成熟、增殖、活化和分化。 來自于LAT基因敲除小鼠實驗模型的研究結果證明LAT分子在T細胞發(fā)育中的關鍵作用， LAT分子缺失的T細胞系的研究顯示LAT分子在T細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細胞中l(wèi)at小鼠的研究結果提示在生理情況下LAT分子在成熟T細胞中表現出和胸腺發(fā)育過程中不同的生物學功能，，主要表現為TCR下游信號的衰減和淋巴細胞的異常增殖和分化。為此，我們推測LAT分子在外周成熟T細胞中還具有不為所知的新的調節(jié)T細胞功能作用模式，并對此進行了本項目的研究。 在本研究中，我們通過Amaxa電轉體系將LAT特異性小片段干擾RNA經體外轉染人外周成熟T細胞，建立瞬時高效干擾LAT分子表達系統(tǒng)，獲得LAT分子缺失原代T細胞和T細胞系，繼而開展功能試驗探討LAT分子對T細胞功能的調節(jié)作用。在成功干擾LAT蛋白的表達之后，我們首先檢測了T細胞增殖以及細胞因子分泌等表型，結果顯示LAT分子缺失的T細胞在TCR/CD28雙信號刺激下，T細胞的增殖能力雖然沒有顯著改變，但是其IL-2的分泌較對照組有顯著的升高。同時，研究結果顯示IFN-的mRNA水平和分泌量則較對照組有所降低，IL-4的mRNA水平升高，提示LAT缺失的T細胞經TCR/CD28活化后具有向Th2細胞分化的趨勢。 為了進一步探討LAT分子缺失后導致的T細胞細胞應答格局改變的分子機制，我們進一步分析TCR介導的信號傳導通路中相關重要信號分子的磷酸化表達譜的改變。結果顯示，在LAT分子缺失的原代T細胞經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，并導致鈣離子內流的缺失；同時Erk1/2活化程度顯著降低。相反，研究結果發(fā)現LAT敲除后的T細胞經TCR/CD28活化過程中，胞內3，4，5三磷酸磷脂酰肌醇(PIP3)出現大量的聚集，該分子是CD28分子參與PI3K/AKT信號通路重要的活化信使分子，從而導致PI3K/AKT信號通路的增強和轉錄因子NF-B的持續(xù)磷酸化。 綜合上述研究結果，我們認為LAT分子在外周成熟T細胞還可能發(fā)揮調節(jié)其他信號通路如CD28信號通路的作用，其中PIP2可能是參與TCR信號途徑與CD28信號途徑轉換的關鍵鏈接分子，在正常表達LAT分子的T細胞中，來自于TCR/CD28的信號可以促進PIP2在PLC-1的作用下水解為IP3和DAG，傳遞正常的TCR活化信號；而在LAT分子缺失的情況下，胞內PIP2則向PIP3轉換，從而增強CD28介導的PI3K/AKT信號通路，以旁路激活的方式介導T細胞功能的改變，上述研究結果首次證實了LAT分子在成熟T細胞中的新的調節(jié)模式，為其所參與的TCR信號轉導通路參與調節(jié)其他信號通路提供了直接證據。
[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 .
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392

【參考文獻】

相關期刊論文 前1條

1 朱香清;陳剛;;PKB/Akt-NF-κB通路與糖尿病血管內皮細胞功能的調節(jié)[J];國際內科學雜志;2007年10期

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