斑马鱼Dock2基因调控T细胞发育机理的研究

Dock2 mutation in patient would lead to immune deficiency disorder. The phenotype includes reduced T cell number and defective immune response. It usually requires hematopoietic stem cell transplantation (HSCT) to cure the disease. However, HSCT is limited by difficulties including donor origin and HLA matching. Understanding the mechanism behind the Dock2 function in T cell development and establishing proper animal model of Dock2 immune deficiency would be valuable for understanding the progress of disease and facilitating the medical trial. .Zebrafish is an excellent animal model for both basic research and medical application. We have generated the Dock2 zebrafish mutant in which several abnormal phenotypes were observed during early T cell development: 1. The expression of rag1, which is essential for V(D)J recombination of T cell receptor (TCR), is partially reduced; 2. The number of T cell/thymocytes is increased; 3. The movement of T cell/thymocytes is reduced. We propose that the mutant could be utilized to study the mechanism of Dock2 function in T cell development and to establish the Dock2 immune deficiency disease model..Here, we propose the following research: 1. Study the cellular mechanism behind the T cell defect in the Dock2 mutant. 2. Unravel the molecular mechanism behind the T cell defect in the Dock2 mutant. 3. Establish the Dock2 zebrafish disease model.

Dock2突变会引起表现为T细胞缺少、免疫反应降低等症状的免疫缺陷疾病，这往往需要造血干细胞移植才能根治，而造血干细胞移植又受到供体以及配型等多方面的限制。理解Dock2基因突变对T细胞发育的影响机理，以及建立合适的Dock2免疫缺陷疾病模型有利于研究Dock2免疫疾病的发生发展机理，为测试药物治疗、尝试免疫重建乃至开发基因治疗提供基础与线索。. 斑马鱼是研究早期T细胞发育以及建立免疫缺陷疾病模型的优秀模式动物。我们建立了Dock2的斑马鱼突变体，在该突变体早期发育中T细胞表现异常：1、T受体TCR的V（D）J区重组所必需的rag1基因表达部分降低；2、T/胸腺细胞的数量反而增加；3、动物活体实时成像表明这些T/胸腺细胞在胸腺中的运动明显降低。本项目拟利用该突变体研究Dock2基因对T细胞发育的影响机理以及建立Dock2突变引起的斑马鱼免疫缺陷疾病模型。

Dock2突变会引起表现为免疫细胞功能障碍、免疫反应降低等症状的免疫缺陷疾病，目前没有特效药物，需要造血干细胞移植才能根治，而造血干细胞移植又受到供体以及配型等多方面的限制。理解Dock2基因突变对免疫细胞发育及功能的影响机理，建立合适的Dock2免疫缺陷疾病模型有利于研究Dock2免疫疾病的发生发展机理，为测试药物治疗、尝试免疫重建乃至开发基因治疗提供基础与线索。斑马鱼是研究早期免疫细胞发育、建立免疫缺陷疾病模型以及开展药物筛选的优秀模式动物。我们构建了Dock2的斑马鱼突变体，该突变体中T细胞数目减少且运动能力下降，中性粒细胞运动能力下降且对机械损伤和细菌感染的应答减弱。进一步研究表明突变体中性粒细胞运动能力的下降可部分通过激活Rac2拯救。我们通过药物筛选发现异丙喘宁能拯救dock2突变体中性粒细胞运动能力的下降，且能能恢复dock2突变体中性粒细胞对细菌感染的应答。此外，我们发现Dock2的结合蛋白Elmo1的斑马鱼突变体表现出和dock2突变体类似的免疫细胞运动和功能障碍，我们进一步通过实时成像检验了三种新型人类 ELMO1 变体，发现p.E90K (c.268G>A) 和 p.D194G (c.581A>G)，可以有效地恢复 elmo1 突变体中中性粒细胞的运动缺陷，而p.R354X (c.1060C>T)则无法恢复ELMO1的功能。我们的研究提示dock2在斑马鱼中发挥保守的功能，可以作为很好的免疫缺陷疾病模型开展药物筛选和治疗的研究。我们筛选到的异丙喘宁是常用药物，进一步探究异丙喘宁对dock2突变免疫细胞的拯救机理，并在更高等的动物上验证该药物可能为治疗dock2免疫缺陷症提供特效药物。