T细胞活化连接蛋白LAT分子参与外周T细胞活化和分化的调节机制探讨

发布时间：2018-05-26 03:46

本文选题：T细胞活化连接蛋白（LAT） + 细胞功能　；参考：《上海交通大学》2012年博士论文

【摘要】：T细胞活化连接蛋白（LAT）是TCR与MHC-抗原复合物结合后，在T细胞抗原受体（TCR）传递活化信号早期阶段发挥关键作用的跨膜连接蛋白，是ZAP-70等Src家族蛋白酪氨酸激酶的重要作用底物。LAT分子胞内段酪氨酸残基被ZAP-70磷酸化后，可以招募诸多胞内游离分子如Grb2, PLC-γ1，Gads等，一方面被招募的PLC-1被活化后通过水解PIP2产生重要信使分子IP3和DAG，启动钙离子内流和激活Ras-MAPK等途径；同时被招募的Gads可以进一步和SLP-76相结合，参与细胞骨架重塑和整合素活性升高，LAT还可以通过结合Grb2-SOS分子并招募其到细胞膜从而实现对Ras-MAPK途径的活化。上述这些直接或间接被LAT招募后所形成的“LAT信号复合体”（LAT Signalsome）实现了来自于抗原的胞外信号经TCR识别后转化成胞内活化信号，最终实现T细胞的发育、成熟、增殖、活化和分化。来自于LAT基因敲除小鼠实验模型的研究结果证明LAT分子在T细胞发育中的关键作用， LAT分子缺失的T细胞系的研究显示LAT分子在T细胞活化中的重要作用，但是来自于LATY136F突变小鼠和条件性剔除外周T细胞中lat小鼠的研究结果提示在生理情况下LAT分子在成熟T细胞中表现出和胸腺发育过程中不同的生物学功能，，主要表现为TCR下游信号的衰减和淋巴细胞的异常增殖和分化。为此，我们推测LAT分子在外周成熟T细胞中还具有不为所知的新的调节T细胞功能作用模式，并对此进行了本项目的研究。在本研究中，我们通过Amaxa电转体系将LAT特异性小片段干扰RNA经体外转染人外周成熟T细胞，建立瞬时高效干扰LAT分子表达系统，获得LAT分子缺失原代T细胞和T细胞系，继而开展功能试验探讨LAT分子对T细胞功能的调节作用。在成功干扰LAT蛋白的表达之后，我们首先检测了T细胞增殖以及细胞因子分泌等表型，结果显示LAT分子缺失的T细胞在TCR/CD28双信号刺激下，T细胞的增殖能力虽然没有显著改变，但是其IL-2的分泌较对照组有显著的升高。同时，研究结果显示IFN-的mRNA水平和分泌量则较对照组有所降低，IL-4的mRNA水平升高，提示LAT缺失的T细胞经TCR/CD28活化后具有向Th2细胞分化的趋势。为了进一步探讨LAT分子缺失后导致的T细胞细胞应答格局改变的分子机制，我们进一步分析TCR介导的信号传导通路中相关重要信号分子的磷酸化表达谱的改变。结果显示，在LAT分子缺失的原代T细胞经TCR/CD28激活后，位于LAT上游的Lck分子的磷酸化没有改变，但位于LAT分子下游的PLC-γ1的磷酸化水平明显下降，并导致钙离子内流的缺失；同时Erk1/2活化程度显著降低。相反，研究结果发现LAT敲除后的T细胞经TCR/CD28活化过程中，胞内3，4，5三磷酸磷脂酰肌醇(PIP3)出现大量的聚集，该分子是CD28分子参与PI3K/AKT信号通路重要的活化信使分子，从而导致PI3K/AKT信号通路的增强和转录因子NF-B的持续磷酸化。综合上述研究结果，我们认为LAT分子在外周成熟T细胞还可能发挥调节其他信号通路如CD28信号通路的作用，其中PIP2可能是参与TCR信号途径与CD28信号途径转换的关键链接分子，在正常表达LAT分子的T细胞中，来自于TCR/CD28的信号可以促进PIP2在PLC-1的作用下水解为IP3和DAG，传递正常的TCR活化信号；而在LAT分子缺失的情况下，胞内PIP2则向PIP3转换，从而增强CD28介导的PI3K/AKT信号通路，以旁路激活的方式介导T细胞功能的改变，上述研究结果首次证实了LAT分子在成熟T细胞中的新的调节模式，为其所参与的TCR信号转导通路参与调节其他信号通路提供了直接证据。
[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 - 纬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 - 纬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 .
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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