大鼠1型糖尿病致病易感基因Vβ13的研究

Type 1 diabetes (T1D) is a T cell mediated autoimmune disorder and characterizes as β cell damages in islets. Factors as genetic, environmental and infective viral ones are considered to be involved compliatly. The fundamental cause of which is unknown. We have observed previously that Iddm14 is a major diabetes susceptibility locus in BB rats and LEW.1WR1 rats amd Vβ13 is a T1D susceptibility gene underlying Iddm14. In this reserch, a cytomegalovirus induced T1D rat model will be set up as a platform. Our aim in this study is to clarify that TCR Vβ13 underlying Iddm14 locus is a key susceptible gene caused by CMV infection by monclonal antibody of Vβ13 blocking protein intraperitoneally injections, adoptive transfer after RNA silencing Vβ13 and constrcting transgenic animal with Vβ13 knocking out.Immuno-precipitation will be used to detect autoantibodies and light microscope as well as electron microscope will be applied to observe the T1D pathological changes and characteristics. By using flow cytometry, RT-PCR and Western Blot, respectively, the expression changes in Vbeta positive cell rate, mRNA and protein expression will be deteced by means of protein level, cell level and animal level, so as to provide theory and experimental basis on revealing T1D pathogenesis and treatment of T1D targets.

1型糖尿病是T淋巴细胞介导的以胰岛β细胞损伤为特征的自身免疫性疾病，该疾病是由遗传因素、环境因素和病毒感染等因素的复杂相互作用共同参与引发的，迄今为止，其发生机理尚未清楚。申请人前期工作中发现Vβ13可能是病毒致1型糖尿病的重要易感基因。本研究在巨细胞病毒感染大鼠模型的基础上，通过大鼠腹腔内注射抗Vβ13单克隆抗体实验、沉默Vβ13的T细胞过继转输实验和转基因大鼠实验，应用流式细胞术、RT- PCR和Western Blot等技术分别检测Vβ13在数量比例、mRNA和蛋白水平的表达变化，分别从蛋白水平、细胞水平和动物水平阐明Iddm14基因座的Vβ13基因是病毒致1型糖尿病的重要易感基因，为揭示1型糖尿病的发病机理和治疗药物的靶点选择提供理论基础和实验依据。

T细胞受体（TCR）Vβ13及其相关炎性因子在1型糖尿病的发生中发挥了重要的作用。本课题通过基于LEW.1WR1-WT大鼠及敲除Vβ13、IFN-I基因鼠为研究对象，结合运用生物信息学、细胞生物学、基因芯片等方法与技术，对Vβ13和IFN参与1型糖尿病发生发展的机制进行了初步研究。首先通过抗体阻断、过继性转移和基因敲除技术，分别从蛋白水平、细胞水平和动物水平验证了Vβ13对于糖尿病发生的重要性。进而深入研究，发现了Treg细胞与Vβ13的密切关系；经基因芯片分析，确定了IFN在T1D发生的重要意义，结果显示 Ifnar1-/-大鼠经KRV感染后1型糖尿病发病率降低，炎性因子CXCL10和CCL5表达均减少。此外，我们在HCMV感染导致其他组织如内皮细胞、神经细胞病变机制的工作正在进行。综合上述结果，本研究为探讨CMV和KRV导致1型糖尿病的机制奠定了坚实的工作基础，对于认识糖尿病的发生与其环境提供了重要线索。项目资助发表SCI论文9篇，核心期刊论文4篇。围绕本项目申请专利3项，获2018年度山东省高等学校科学技术二等奖1项。培养硕士生5名，其中2名已经取得硕士学位，3名在读。项目投入经费73万元，支出73万元，各项支出基本与预算相符。