肠道免疫反应引起的还原性微环境对淋巴细胞黏附与迁移的调控及其机制研究

Cysteine/cystine (Cys/Cyss) redox system has been recently proposed as a major antioxidant mechanism. In inflammatory bowel disease (IBD), T lymphocytes activation and proliferation rely on a reducing extracellular environment that is provided by antigen presenting cells, which secrete a large number of cysteine. L-Cysteine is the only amino acid that has an important functional free –SH group, which is required for glutathione (GSH) synthesis in T lymphocytes. Thus the amount of free thiols increases dramatically during the immune response in lymphoid tissues. Our previous studies have shown that the reducing agent-dithiothreitol can activate integrin α4β7, which plays critical roles in mediating lymphocytes homing to the gut associated lymphoid tissues (GALT). However, the mechanism of the reducing microenvironment induced by the immune response in regulating lymphocyte adhesion and transmigration is still obscure. In the present study, we demonstrated for the first time that cysteine can activate integrin α4β7 in vitro, as well as influences integrin α4β7 mediated cell adhesion and migration. Our study also showed that the disulfide bonds located at the extracellular domain of integrin α4β7 could be reduced by cysteine. Based on these data, we plan to further investigate the molecular mechanism of integrin α4β7 activation by cysteine, and test the mechanism underlying the regulation of lymphocytes migration by reducing microenvironment using mouse model. At the same time, we will explore the clinical significance of the correlation between the reducing state in intestinal tissue and the activity of integrin α4β7 in inflammatory bowel disease. These studies will reveal a novel mechanism for the regulation of lymphocytes migration and provide a new reference for disease treatment.

半胱氨酸/胱氨酸（Cysteine/Cystine）氧化还原系统是主要的细胞外抗氧化机制。在炎症性肠病（IBD）中，抗原呈递细胞分泌大量cysteine供给T淋巴细胞，肠道组织中自由巯基的含量明显增加。前期研究发现二硫苏糖醇等还原剂可以活化整合素α4β7，而后者的活化可以介导淋巴细胞异常浸润到肠道引起炎症反应。目前对还原性微环境调控淋巴细胞黏附迁移的机制还不清楚。在本项目的研究中我们首次在体外证明cysteine可以活化整合素α4β7并影响其介导的细胞黏附迁移，还发现cysteine可以还原整合素α4β7胞外段分子内部的二硫键。在此基础上，我们将深入研究cysteine是否通过由外而内的信号通路调控整合素α4β7的活化，并且利用小鼠模型进一步研究肠道组织中还原性微环境对整合素活性及其介导的淋巴细胞迁移的全新调控机制。同时我们将探究此调控机制的临床意义，为炎症性肠病的治疗提供新的思路。

在本项目的研究中我们首次在体外发现cysteine可以活化整合素α4β7并影响其介导的细胞黏附迁移，证明了cysteine活化α4β7具有浓度和时间的依赖性。随后我们深入研究了cysteine调控α4β7活化的机制并且发现cysteine主要通过还原α4β7胞外段分子内部的二硫键由外而内活化整合素并且影响下游FAK的磷酸化水平。在此基础上，我们建立了小鼠炎症性肠病以及肠癌模型，检测发现在两种小鼠的肠道组织中有不同程度的自由巯基增加。同时分别在两种小鼠的结肠和直肠组织处发现有CD4+T细胞的浸润增加以及α4β7的配体MAdCAM-1的表达增加，由此间接证明了α4β7表达与自由巯基的相关性。目前我们正在重复小鼠模型实验进一步检测α4β7在组织中的表达水平，并且检测cysteine处理对小鼠淋巴细胞的体内归巢影响，以期直接证明cysteine对α4β7介导的淋巴细胞迁移的影响。我们也已经搜集一部分临床样本，希望能够在临床水平进一步揭示两者的相关性。.另外，本项目在研究肠炎和肠癌进展关系的过程中，意外发现整合素α5在肠癌组织中表达明显升高，并且α5主要表达在肠道肿瘤基质而不是上皮细胞上。以往关于α5的报道主要集中在肠癌细胞中，因而我们对整合素α5在肠癌间质中的表达意义进行了深入研究。我们首先证实α5主要在癌症相关的成纤维细胞中表达。裸鼠实验表明α5表达对于对成纤维细胞的促肿瘤作用是必需的。在体外细胞共培养系统中，α5的敲除降低了成纤维细胞促进癌细胞迁移和侵袭的能力；此外我们观察到α5敲除后纤连蛋白的表达和装配被下调。TCGA数据表明ITGA5（α5整合素亚基）与大肠腺癌总体生存率低相关，这一结果在我们自己搜集的包含有355名中国大肠腺癌患者独立组中进一步得到证实。因此，我们的研究鉴定了α5整合素亚基能够作为结直肠腺癌的新型基质分子标记，提供了有关结直肠腺癌进展的新见解。