【作者】 龚燕萍；

【导师】 熊思东； 张瑞华； 徐薇； 储以微；

【作者基本信息】 复旦大学 ， 免疫学， 2007， 博士

【摘要】 胸腺是T淋巴细胞发育的重要场所，为T细胞发育提供了一个特定的微环境。胸腺T细胞发育、分化和功能成熟的研究是当今免疫学研究的重要领域之一。骨髓来源的Pro-T细胞通过血液循环迁入胸腺。Pro-T细胞表型为CD4~-CD8~-(double negative，DN)。Pro-T细胞在胸腺微环境包括胸腺基质细胞、细胞外基质和细胞因子等多种因素的精确调控下，发育成为CD4~+CD8~+双阳性(double positive，DP)细胞；DP细胞再经阳性选择(positive selection)和阴性选择(negative selection)发育成具有自身耐受性、自身MHC限制性和抗原特异性的成熟的CD4~+或CD8~+单阳性(single positive，SP)细胞，输出到外周执行免疫应答功能。T细胞发育、胸腺选择过程的调节机制仍未完全阐明。目前认为，DP细胞上TCR与胸腺上皮细胞上自身MHC／自身肽复合物的相互作用对T细胞发育的命运起决定性作用，而其它受体—配体分子也对TCR的信号转导和胸腺选择产生影响。近年来的研究发现DP细胞和胸腺上皮细胞的相互作用过程中形成了与外周成熟T细胞相似的免疫突触，并且干预参与免疫突触的相关分子可以通过影响其突触形成而最终影响其分化发育的结果。肌萎缩蛋白(Dystroglycan，DG)是DGC(dystrophin glycoprotein complex)的重要组成部分，是连接细胞外基质和细胞骨架蛋白的重要分子。DG由α和β两个亚基组成，α-DG为胞膜外部分，是组成DGC复合物的中心蛋白成分，可以和多种胞外基质蛋白结合，如agrin，perlecan，laminin，merosin，neurexin，biglycan等；β-DG为跨膜及细胞内部分，通过其胞内段的SH3功能结合域与细胞内的dystrophin或utrophin结合或直接与actin结合，使细胞外基质蛋白与细胞骨架蛋白相联结。DG被证实参与了神经肌接头突触的形成。Campbell等人的研究发现Dystroglycan的缺失导致早期胚胎由于缺乏Reichert氏膜而死亡，这一结果提示Dystroglycan在胚胎的早期存活中具有极其重要作用。除了肌肉组织以外，DG还广泛表达在许多非肌肉组织如脑、肾脏、肝脏、消化道和气管等中。作为基质蛋白laminin和agrin等的受体，DG在非肌肉组织中参与一些生理和病理过程，比如上皮的发生和肿瘤的生成等，其作用特点是与基质蛋白相互作用从而参与细胞的发育、增殖等。在胸腺发育方面，DG的一个配体laminin-2和其integrin receptor VLA-6的相互作用为胸腺细胞的发育提供生存信号和分化信号。laminin-2 Knock Out小鼠胸腺大小较正常小鼠小，胸腺细胞数目较正常小鼠少，胸腺的结构也发生了变化。laminin-5是DG的另一个配体，胸腺基质细胞上laminin-5与胸腺细胞的相互作用是胸腺细胞生存和分化所必需的。DG的另一个配体merosin也在胸腺中有表达并且为胸腺细胞的增殖提供共刺激信号。细胞外基质蛋白与其受体的相互作用相当复杂，DG的众多配体在胸腺中都有表达且对胸腺细胞发育起到一定的作用，那么DG在胸腺细胞中是否有表达，其在胸腺细胞发育中是否起到一定的作用呢?我们前期的研究已经发现α-DG广泛表达在各种免疫细胞中，并参与了外周成熟T细胞的活化，且具有共刺激分子样作用，提示我们α-DG也可能在胸腺细胞中表达并起到一定的作用。据此本研究对α-Dystroglycan在胸腺细胞中的分布和表达、在胸腺细胞发育中的作用、以及其作用机制进行了研究。第一部分α-Dystroglycan在胸腺细胞中动态表达的体内外研究一．α-Dystroglycan在不同日龄胎鼠胸腺中动态表达为研究α-Dystroglycan是否在胸腺中有表达，首先用RT-PCR检测发现在孕15天～孕19天胎鼠的胸腺中均有α-Dystroglycan的表达；从半定量的结果来看，α-Dystroglycan mRNA在孕15天～孕16天胎鼠的胸腺中的表达呈现一个上升趋势，在孕16天达到高峰，之后在孕16天～孕19天逐渐呈现一个下降趋势。这部分结果表明α-Dystroglycan动态性地表达于不同日龄胎鼠胸腺中，为之后的研究提供了基础。二．α-Dystroglycan在体内不同日龄胎鼠的各亚群胸腺细胞中动态表达为研究α-Dystroglycan在体内胸腺细胞中的表达情况，我们应用FACS进一步从蛋白水平检测发现：α-Dystroglycan广泛分布在体内不同日龄胎鼠的各亚群胸腺细胞中，特别是CD4~+CD8~+阳性细胞和CD8~+／CD4~+单阳性细胞：以孕19天胎鼠为例，α-Dystroglycan在总体胸腺细胞、CD4~-CD8~-双阴性细胞、CD4~+CD8~+双阳性细胞、CD4~+单阳性细胞和CD8~+单阳性细胞上的表达分别为92.66％、8.30％、99.98％、90.42％和95.41％。另外就单独的一个胸腺细胞亚群来看，α-Dystroglycan在体内孕15天～孕19天胎鼠的同一胸腺细胞亚群中的表达也呈现出动态的特点，特别是对CD4~+单阳性细胞和CD8~+单阳性细胞来说，α-Dystroglycan的表达就呈现一个相对上升的趋势，CD4~+单阳性细胞在孕16天～孕19天的表达分别是54.55％，80.22％，76.60％，90.42％，CD8~+单阳性细胞在孕16天～孕19天的表达分别是52.34％，50.35％，62.71％，95.41％；而对CD4~+CD8~+双阳性细胞来说，α-Dystroglycan的表达就相对稳定在一个较高的水平，在孕16天～孕19天的表达分别是97.62％，99.57％，99.59％，99.98％。此结果说明α-Dystroglycan动态性地表达于体内不同日龄胎鼠的各亚群胸腺细胞中，也提示了α-Dystroglycan可能在胸腺细胞发育中起到一定的作用。三．α-Dystroglycan在体外培养不同天数的各亚群胸腺细胞中动态表达为研究α-Dystroglycan在体外胎鼠胸腺器官培养(FTOC)系统培养的胸腺细胞中的表达情况，首先我们建立FTOC系统，然后我们应用FACS进一步从蛋白水平检测发现：与体内相似的，α-Dystroglycan广泛分布在体外培养2～6天的各亚群胸腺细胞中，以FTOC体外培养6天的胎鼠胸腺细胞为例，α-Dystroglycan在总体胸腺细胞、CD4~-CD8~-双阴性细胞、CD4~+CD8~+双阳性细胞、CD4~+单阳性细胞和CD8~+单阳性细胞上的表达分别为85.35％、73.07％、87.06％、98.09％和93.47％，可见α-Dystroglycan在双阳性细胞和单阳性细胞上表达较丰富。另外就单独的一个胸腺细胞亚群来看，α-Dystroglycan的表达在FTOC体外培养2～6天的同一胸腺细胞亚群中的表达也呈现出动态的特点，即表现出一个相对上升的趋势。此结果与体内的结果相似，说明α-Dystroglycan同样也动态性地表达于体外培养不同天数的各亚群胸腺细胞中。第一部分的研究结果表明α-Dystroglycan动态性地表达于体内不同日龄胎鼠的各亚群胸腺细胞中以及体外培养不同天数的各亚群胸腺细胞上，特别是较丰富地表达在CD4~+CD8~+双阳性细胞和CD8~+／CD4~+单阳性细胞上，这部分研究为之后的研究提供了基础。第二部分α-Dystroglycan在胸腺细胞发育中的作用一．α-Dystroglycan在双阴性细胞发育中的作用第一部分的结果表明α-Dystroglycan较高地表达于双阳性细胞以及单阳性细胞中，但在双阴性细胞中表达则相对较低，部分提示我们α-Dystroglycan有可能在双阳性细胞以及单阳性细胞发育中起到一定的作用，而在双阴性细胞发育中起作用的可能性则不大。为研究α-Dystroglycan是否在双阴性细胞发育中起作用，我们在FTOC中加入抗α-Dystroglycan的阻断性抗体，在蛋白质水平阻断其作用来观察对双阴性细胞分化发育的影响，结果发现：与未经任何处理对照组和同型抗体对照组相比，抗α-Dystroglycan的阻断性抗体组中双阴性细胞的绝对数未发生显著变化。此结果提示α-Dystroglycan在双阴性细胞的发育中可能不起作用。为进一步验证α-Dystroglycan在双阴性细胞的发育中不起作用，同时我们还建立胸腺器官重构培养即RTOC系统，然后在RTOC中引入携带反义Dystroglycan基因的逆转录病毒，在基因水平下调其表达及作用来观察对双阴性细胞分化发育的影响，结果发现：与无病毒组和对照空病毒组相比，携带反义Dystroglycan基因的逆转录病毒组中双阴性细胞的绝对数未发生显著变化。此结果与抗体阻断的结果相似，也提示α-Dystroglycan在双阴性细胞的发育中可能不起作用。二．α-Dystroglycan在双阳性细胞发育中的作用α-Dystroglycan高表达于双阳性细胞中，提示我们α-Dystroglycan很有可能在双阳性细胞发育中起到一定的作用。为研究α-Dystroglycan是否在双阳性细胞发育中起到一定的作用，我们在FTOC中加入抗α-Dystroglycan的阻断性抗体，在蛋白质水平阻断其作用来观察对双阳性细胞分化发育的影响，结果发现：与未经任何处理对照组和同型抗体对照组相比，抗α-Dystroglycan的阻断性抗体能明显抑制双阳性细胞分化发育，表现为双阳性细胞比例明显减少，从对照组的39.81％降到抗体干预组的7.5％；双阳性细胞绝对数也显著减少。而同型对照组和未处理对照组比差异均不显著。此结果说明阻断α-Dystroglycan抑制了双阳性细胞的发育，也提示α-Dystroglycan在双阳性细胞发育中起到一定的作用。为进一步验证α-Dystroglycan在双阳性细胞的发育中确实起作用，同时我们在RTOC中引入携带反义Dystroglycan基因的逆转录病毒，在基因水平下调其表达及作用来观察对双阳性细胞分化发育的影响，结果发现：培养6天后，PA317-pLXSN-AS-DG病毒组总体胸腺细胞和双阳性细胞的α-Dystroglycan的表达分别为7.90％和53.12％，均较PA317组30.98％和87.34％降低，而PA317-pLXSN空病毒组仍保持在30.26％和81.91％。此结果说明PA317-pLXSN-AS-DG所产生的重组病毒可以在本RTOC系统中抑制发育中的胸腺细胞包括双阳性细胞上α-Dystroglycan的表达，而对照的PA317-pLXSN则无类似作用。观察重组病毒对双阳性细胞发育的影响发现下调双阳性细胞上Dystroglycan的表达能明显抑制双阳性细胞分化发育，表现为双阳性细胞比例显著下降，由对照组的20.61％下降到1.84％；双阳性细胞绝对数也显著减少。而PA317-pLXSN组双阳性细胞的比例和绝对数均与PA317对照组无显著差异。此结果与抗体的结果相似，说明下调α-Dystroglycan抑制了双阳性细胞的发育，也进一步提示α-Dystroglycan在双阳性细胞发育中起到一定的作用。三．α-Dystroglycan在单阳性细胞发育中的作用α-Dystroglycan较高地表达于单阳性细胞中，提示我们α-Dystroglycan也有可能在单阳性细胞发育中起到一定的作用。为研究α-Dystroglycan是否在单阳性细胞发育中起到一定的作用，我们在FTOC中加入抗α-Dystroglycan的阻断性抗体，在蛋白质水平阻断其作用来观察对单阳性细胞分化发育的影响，结果发现：与未经任何处理对照组和同型抗体对照组相比，抗α-Dystroglycan的阻断性抗体组中CD8~+单阳性细胞比例则显著下降，从对照组的20.66％下降到抗体干预组的6.84％，而CD4~+单阳性细胞比例无显著变化；但CD4~+和CD8~+单阳性细胞的绝对数都显著下降；而同型对照组和未处理对照组比各项差异均不显著。同时我们在RTOC中引入携带反义Dystroglycan基因的逆转录病毒，在基因水平下调其表达及作用来观察对单阳性细胞分化发育的影响，结果发现：与无病毒组和对照空病毒组相比，携带反义Dystroglycan基因的逆转录病毒组中CD4~+和CD8~+单阳性细胞的比例和绝对数均表现出下降。此结果与抗体阻断的结果相似。由于干预α-Dystroglycan同时抑制了双阳性细胞和单阳性细胞的发育，提示α-Dystroglycan可能在双阳性细胞和单阳性细胞发育中起到一定的作用，也有可能对单阳性细胞的影响是对双阳性细胞直接影响的后续结果。第二部分的研究结果表明阻断或下调α-Dystroglycan对双阴性细胞的发育没有影响，但抑制了双阳性细胞和单阳性细胞的发育，提示α-Dystroglycan在双阴性细胞发育中可能不起作用，而在双阳性细胞发育中起到一定的作用，在单阳性细胞发育中可能起到一定的作用，也有可能对单阳性细胞的影响是对双阳性细胞直接影响的后续结果。第三部分α-Dystroglycan在胸腺细胞发育中的作用机制一．α-Dystroglycan对胸腺细胞凋亡的影响干预α-Dystroglycan抑制了胸腺细胞的发育，主要表现为细胞数目的减少，那么凋亡是否是其数目减少的原因呢?为探讨α-Dystroglycan在胸腺细胞发育中的作用机制，我们检测了阻断α-Dystroglycan对胸腺细胞凋亡的影响：首先通过检测细胞亚二倍体峰发现抗体干预组的胸腺细胞总体凋亡水平明显增加，由25.90％上升到45.82％；而同型对照组和未处理对照组相比差异不显著；进一步用7-AAD检测各亚群胸腺细胞的凋亡情况，结果发现：与对照组相比，抗体干预组的双阳性细胞凋亡明显增加，由52.84％上升到97.83％，同时发现单阳性细胞凋亡也有一定程度增加，而双阴性细胞凋亡则无显著变化，而同型对照组和未处理对照组相比差异不显著。以上结果说明阻断α-Dystroglycan促进了胸腺细胞特别是双阳性细胞的凋亡，也提示α-Dystroglycan可能为胸腺细胞特别是双阳性细胞提供抗凋亡和(或)生存信号。为此我们进一步检测了α-Dystroglycan对双阳性细胞凋亡的影响。我们将分离纯化的双阳性细胞与胸腺上皮细胞以1：1比例混合进行RTOC培养，结果发现：在培养后的36小时，与对照组相比，抗体干预组双阳性细胞的凋亡明显增加，由62.28％上升到94.55％；与此相一致，在培养后的第3天，与对照组相比，抗体干预组CD4~+单阳性细胞和CD8~+单阳性细胞的比例和数目也显著降低。此结果说明阻断α-Dystroglycan促进了胸腺细胞特别是双阳性细胞的凋亡和阳性选择的减少，也提示α-Dystroglycan可能为胸腺细胞特别是双阳性细胞提供抗凋亡和(或)生存信号。二．α-Dystroglycan在胸腺细胞和胸腺上皮细胞间免疫突触形成中的作用有研究提示免疫突触的形成对胸腺细胞发育过程中所需的生存信号非常重要，影响免疫突触形成的因素可能会影响胸腺细胞的生存信号并最终影响其发育结果。因此，我们推测α-Dystroglycan可能通过参与胸腺细胞免疫突触的形成为其提供正常发育所必需的生存信号，阻断α-Dystroglycan可能通过影响免疫突触的形成从而影响胸腺细胞的生存信号，并最终影响其发育结果。为探讨α-Dystroglycan是否在胸腺细胞和胸腺上皮细胞间免疫突触形成中起作用，我们观察了阻断α-Dystroglycan对胸腺细胞和胸腺上皮细胞间免疫突触形成的影响：首先纯化双阳性细胞并建立体外双阳性细胞和胸腺上皮细胞相互作用系统，在此基础上，观察抗α-Dystroglycan的阻断性抗体对双阳性细胞和胸腺上皮细胞相互作用的影响，结果发现：与对照组相比，双阳性细胞和胸腺上皮细胞结合体形成减少，抗体组细胞骨架蛋白重排减少。此结果提示阻断α-Dystroglycan抑制了双阳性细胞和胸腺上皮细胞间免疫突触的形成，也提示α-Dystroglycan在双阳性细胞和胸腺上皮细胞间免疫突触的形成过程中起到一定的作用。三．α-Dystroglycan影响胸腺细胞凋亡的可能途径CD95／CD95L途径被证实在胸腺细胞的凋亡中起到一定的作用。而Bcl-2的上调表达与胸腺细胞的阳性选择紧密相关。为探讨在这一模型中α-Dystroglycan影响胸腺细胞凋亡的可能途径，我们用FACS检测了胸腺细胞CD95和CD95L的表达，结果发现：与对照组相比，抗体干预组的双阳性细胞CD95的表达明显增加，由53.37％上升到83.05％，同时单阳性细胞CD95的表达也有一定程度增加，而双阴性细胞则无显著变化；相似的，抗体干预组的双阳性细胞CD95L的表达明显增加，同时单阳性细胞CD95L的表达也有一定程度增加，而双阴性细胞则无显著变化；而同型对照组和未处理对照组CD95和CD95L的表达差异均不显著。另外，用Real-time PCR检测胸腺细胞Bcl-2／Bax的表达结果发现：与对照组相比，抗体干预组的Bcl-2的表达显著下降，而Bax的表达则无明显变化。此结果提示α-Dystroglycan可能通过CD95／CD95L和Bcl-2两条途径影响胸腺细胞特别是双阳性细胞的凋亡。以往的研究提示α-Dystroglycan通过MAKP／ERK途径提供肌细胞生存信号，而在胸腺发育中ERK的磷酸化是双阳性细胞获得足够的生存信号并被阳性选择的关键。我们推测α-Dystroglycan可能部分通过MAKP／ERK信号途径提供双阳性细胞生存信号，而阻断α-Dystroglycan可能部分通过影响MAKP／ERK途径下调了双阳性细胞的生存信号从而导致其凋亡及阳性选择的减少。为了明确这一点，我们将分离纯化的双阳性细胞与胸腺上皮细胞进行RTOC培养，在培养后的36小时FACS检测抗体组和对照组双阳性细胞ERK1／2的磷酸化情况，结果发现：与对照组相比，抗体组双阳性细胞ERK1／2的磷酸化情况明显减少，由38.46％下降到16.54％。此结果说明阻断α-Dystroglycan可能部分通过影响MAKP／ERK途径下调了双阳性细胞的生存信号从而导致其凋亡，也提示α-Dystroglycan可能部分通过MAKP／ERK途径提供双阳性细胞生存信号。第三部分的研究结果表明阻断α-Dystroglycan促进了胸腺细胞特别是双阳性细胞的凋亡和阳性选择的减少。α-Dystroglycan通过参与CD4~+CD8~+阳性细胞和胸腺上皮细胞间免疫突触的形成来影响CD4~+CD8~+阳性细胞生存和凋亡信号及最终发育结果。干预α-Dystroglycan影响双阳性细胞和胸腺上皮细胞间免疫突触的形成，并可能部分通过MAPK／ERK途径下调双阳性细胞的生存信号，通过CD95／CD95L和Bcl-2两条途径促进双阳性细胞凋亡，最终影响其发育结果。综上所述，我们的结果提示α-Dystroglycan广泛且动态性地表达在胸腺细胞中。阻断α-Dystroglycan促进了胸腺细胞特别是双阳性细胞的凋亡和阳性选择的减少，也提示α-Dystroglycan可能为胸腺细胞特别是双阳性细胞提供抗凋亡和(或)生存信号。α-Dystroglycan通过参与双阳性细胞和胸腺上皮细胞间免疫突触形成为双阳性细胞提供正常发育所需的生存信号，干预α-Dystroglycan可能通过影响双阳性细胞和胸腺上皮细胞间免疫突触的形成影响双阳性细胞的生存信号，并促进其发生凋亡，最终影响其发育结果。更多还原

【Abstract】 The task of generating a T lymphocyte population that responds to foreignpeptides presented by MHC, but not to self peptides, is undertaken in thethymus. Thymocytes comprising the newly formed TCR repertoire are selectedfor their ability to recognize peptide in the context of MHC molecules. Positiveselection tests the ability of TCR to signal in response to self-peptide/MHC.Thymocytes that do not receive a TCR signal of sufficient strength die throughneglect. Negative selection eliminates thymocytes expressing TCRs thattransmit a strong signal in response to self-peptide/MHC. Current modelsemphasize the role of overall signal strength, reflecting the input fromcostimulatory and accessory molecules as well as TCR, in determining thedevelopmental outcome of TCR signaling. In mature T cells, TCR signaling isassociated with the formation of immunological synapse, at the T cell/APCinterface. Recent studies have begun to analyze immunological synapseformation during thymic selection using lipid bilayers containing peptide/MHCcomplexes, a negative selection system and a positive selection system.These studies provide direct evidence that thymocyte-epithelial cellinteractions leading to positive selection result in the redistribution of cellmembrane-associated signaling molecules to the thymocyte-epithelial cellinterface in a manner analogous to that seen in mature T cell-APC interactions.Dystroglycan consists ofαandβsubunits yielded by proteolysis cleavage of asingle precursor protein,α-Dystroglycan is a highly glycosylated extracellularprotein and non-covalently anchored to the transmembraneβ-dystroglycan.(α-Dystroglycan links to the extracellular matrix (ECM) via several ligands,whereasβ-dystroglycan linksα-dystroglycan to the actin cytoskeleton viadystrophin or utrophin. Accumulating evidence indicates that dystroglycan is involved in the formation of synapse in neural-muscle junction (NMJ) andcentral never system (CNS). Our previous studies have shown thatα-dystroglycan is expressed on lymphocytes and suggested thatα-dystroglycan might contribute to lymphocyte activation by participating insynapse formation. In this study we found thatα-dystroglycan was dynamicallyexpressed on various thymocytes especially on double positive thymocytesand showed that interruption ofα-dystroglycan led to increased apoptosis ofdouble positive thymocytes by affecting immune synapse formation, indicatingthatα-dystroglycan might be involved in thymocyte development byparticipating in synapse formation.1. Dynamic expression ofα-dystroglycan on fetal thymocytes in vivo andin fetal thymus organ culture (FTOC)(1) Dynamic expression ofα-dystroglycan in fetal thymuses fromdifferent gestation days mice: To investigate the possible roles ofα-dystroglycan in thymocyte development, the expression ofα-dystroglycan inthymus was first examined at mRNA level. The coding gene for dystroglycancould be specifically amplified from total RNA of thymocytes from 15-19gestation days (gd) fetal mice. And an expression peak appeared at 16 gd.The results suggested thatα-dystroglycan was dynamically expressed in fetalthymuses from different gd mice.(2) Dynamic expression ofα-dystroglycan on four subpopulation ofthymocytes in vivo: To examine which subpoplations of thymocytes expressα-dystroglycan in vivo, thymocytes from 15-19 gd fetal mice were stained withantibodies againstα-dystroglycan followed by FITC-labeled rabbit anti-mouseIgG along with anti-CD4-PerCP and anti-CD8-PE and analyzed by FACS. Theresults showed thatα-dystroglycan was dynamically expressed on foursubpopulation of thymocytes from different gd fetal mice, especially highlyexpressed on double positive (DP) cells and CD4+/CD8+ single positive (SP)cells. For example, the expression rates ofα-dystroglycan on total population,DN, DP, CD4+SP CD8+SP cells from 19 gd fetal mice were 92.66%, 8.30%,99.98%, 90.42% and 95.41% respectively; the expression rates of α-dystroglycan on DP cells from 15-19 gd fetal mice were 97.62%, 99.57%,99.59% and 99.98% respectively; the expression rates ofα-dystroglycan onCD4+SP cells from 15-19 gd fetal mice were 54.55%, 80.22%, 76.60% and90.42% respectively; the expression rates ofα-dystroglycan on CD8+SP cellsfrom 15-19 gd fetal mice were 52.34%, 50.35%, 62.71%, and 95.41%respectively. These results probably suggested a role ofα-dystroglycan inthymocyte development.(3) Dynamic expression ofα-dystroglycan on four subpopulation ofthymocytes in FTOC: To examine which subpoplations of thymocytesexpressα-dystroglycan in FTOC, thymocytes recovered from FTOC culturedfor 2-6 days were stained with antibodies againstα-dystroglycan followed byFITC-labeled rabbit anti-mouse IgG along with anti-CD4-PerCP andanti-CD8-PE and analyzed by FAGS. The results showed thatα-dystroglycanwas dynamically expressed on four subpopulation of thymocytes recoveredfrom FTOC cultured for 2-6 days, especially highly expressed on DP and SPcells. For example, the expression rates ofα-dystroglycan on total population,DN, DP, CD4+SP CD8+SP cells recovered from FTOC cultured for 6 dayswere 85.35%, 73.07%, 87.06%, 98.09% and 93.47% respectively and theexpression rates ofα-dystroglycan on the same subpopulation recovered fromFTOC cultured for 2-6 days also varied dynamically. These results probablysuggested a role ofα-dystroglycan in thymocyte development.2. Role ofα-dystroglycan in thymocyte development(1) Role ofα-dystroglycan in development of double negativethymocytesThe above results showed us that the expression ofα-dystroglycan on DPand SP cells was relatively higher, and the expression ofα-dystroglycan onDN cells was relatively lower, partly suggesting thatα-dystroglycan might playa role in development of DP and SP cells, while the possibility thatα-dystroglycan might play a role in development of DN cells was much lower.To explore whetherα-dystroglycan plays a role in development of DN cells, wetreated FTOC with anti-α-dystroglycan blocking antibody IIH6C4 and found that blockage ofα-dystroglycan had no effect on the proportion and number ofDN cells compared with non-treated or isotype antibody-treated FTOC. Theresults indicated thatα-dystroglycan might not play a role in development ofDN cells.To further confirm the above results from Ab blocking experiment, weestablished reaggregate thymus organ culture (RTOC) with virus-producingcells (VPCs) which produced viruses carrying anti-sense cDNA ofα-dystroglycan and found that viruses carrying anti-sense cDNA ofα-dystroglycan had no effect on the proportion and number of DN cellscompared with no viruses and control viruses. The results further indicated thatα-dystroglycan might not play a role in development of DN cells.(2) Role ofα-dystroglycan in development of double positive thymocytesThe results thatα-dystroglycan was highly expressed on DP partlysuggested thatα-dystroglycan might play a role in development of DP cells. Toexplore whetherα-dystroglycan plays a role in development of DP cells, wetreated FTOC with anti-α-dystroglycan blocking antibody IIH6C4 and foundthat blockage ofα-dystroglycan decreased the proportion and number of DPcells compared with non-treated or isotype antibody-treated FTOC. The resultsindicated thatα-dystroglycan might play a role in development of DP cells.To further confirm the above results from Ab blocking experiment, weestablished reaggregate thymus organ culture (RTOC) with virus-producingcells (VPCs) which produced viruses carrying anti-sense cDNA ofα-dystroglycan and found that downregulation ofα-dystroglycan decreasedthe proportion and number of DP cells compared with no viruses and controlVPCs. The results further indicated thatα-dystroglycan might play a role indevelopment of DP cells.(3) Role ofα-dystroglycan in development of single positive thymocytesThe results thatα-dystroglycan was moderately expressed on SP partlysuggested thatα-dystroglycan might play a role in development of SP cells. Toexplore whetherα-dystroglycan plays a role in development of SP cells, wetreated FTOC with anti-α-dystroglycan blocking antibody IIH6C4 and foundthat blockage ofα-dystroglycan decreased the proportion and number of SP cells to a less extent than that of DP cells compared with non-treated orisotype antibody-treated FTOC.To further confirm the above results from Ab blocking experiment, weestablished reaggregate thymus organ culture (RTOC) with virus-producingcells (VPCs) which produced viruses carrying anti-sense cDNA ofα-dystroglycan and found that downregulation ofα-dystroglycan decreasedthe proportion and number of SP cells to a less extent than that of DP cellscompared with no viruses and control VPCs. These results were similar withthe results from Ab blocking experiments. Interruption ofα-dystroglycaninhibited development of both DP cells and SP cells, indicating thatα-dystroglycan might play a role in development of both DP cells and SP cellsor that the effects ofα-dystroglycan on development of SP cells were thefollowing outcome of the effects ofα-dystroglycan on development of DP cells.3. Mechanisms for the role ofα-dystroglycan in thymocyte development(1) The effect ofα-dystroglycan on apoptosis of thymocytes: Toinvestigate whether apoptosis was the reason that caused the numberdecrease of thymocytes, we examined the apoptosis status of thymocytes inIIH6C4-treated lobes. Enhanced apoptosis was observed in DP cells and to aless extent in SP cells in IIH6C4-treated lobes. The results suggested thatα-dystroglycan might be related with anti-apoptosis and/or survival signals ofthymocytes especially that of DP cells.(2) The role ofα-dystroglycan in synapse formation between thymocytesand thymic epithelial cells: To explore the mechanisms for the role ofα-dystroglycan in development of DP cells, we then investigated whetherα-dystroglycan played a role in synapse formation between DP cells andthymic epithelial cells. Using a thymocyte-epithelial cell conjugate model wefound thatα-dystroglycan blockage reduced conjugate formation and actinrearrangement between DP cells and thymic epithelial cells. These resultsindicated thatα-dystroglycan was involved in synapse formation between DPcells and thymic epithelial cells. (3) The pathways involved in the effect ofα-dystroglycan on apoptosis ofthymocytes: Then we examined the possible pathways that might be involvedin the effect ofα-dystroglycan on apoptosis of thymotyes. FAGS analysisdemonstrated that in IIH6C4-treated lobes, the expression of CD95 andCD95L was preferentially increased in DP cells and to a less extent in SP cellsbut not in DN cells compared to that of non-treated or isotype antibody-treatedlobes. Real-time RT-PCR showed that in IIH6C4-treated lobes, the expressionof Bcl-2 was decreased compared to that of non-treated or isotypeantibody-treated lobes. The results indicated that both CD95/CD95L and Bcl-2pathways were probably involved in the increased apoptosis of thymocytesespecially that of DP cells induced byα-dystroglycan blockage. FAGS analysisalso showed that blockage ofα-Dystroglycan downregulated ERKphosphorylation in DP cells, indicating that ERK pathway might be related toα-Dystroglycan signaling.In summary, our results suggested that interruption ofα-dystroglycan led toincreased apoptosis of DP thymocytes by affecting immunological synapseformation, indicating thatα-dystroglycan might be involved in thymocytedevelopment by participating in synapse formation.更多还原