新的恒河猴自身免疫性糖尿病模型的建立及相关机理研究

In order to further understand the pathogenesis of autoimmune diabetes, and to develop effective treatment, appropriate animal model is critical. Non-obese diabetic (non-obesity diabetes, NOD) mice and biobreeding (BB) rats have been used for the study of autoimmune diabetes. However, the rodent models are very different from human beings.These models can not fully simulate the human disease, can not provide appropriate and sufficient evidence for human autoimmune diabetes. It is difficult to translate the research results to human. Our previous studies using rhesus monkey model of diabetes induced by multiple low dose STZ (MLD-STZ) method, showing the characteristics of autoimmune diabetes. This study intends to further optimize the modeling strategy. We are going to test whether the method of T regular cells (Tregs) deleted by anti-CD25 monoclonal antibody plus MLD-STZ is better and also study the related mechanisms. The induction of autoimmune diabetes was evaluated by regular metabolic testing, serological assessment of islet-reactive autoantibodies, cytokines, and histological examination of pancreatic tissues, spleen cells and lymph nodes. Immunohistochemistry, flow cytometry, cell adoptive periodic and autologous islet transplantation are going to be used to test the experimental methods of treatment, further verify the specific destruction of pancreatic β cells using this method to establish the rhesus monkey model of diabetes is caused by T cell-mediated immune response, rather than the toxic effects of streptozotocin. This will serve a preclinical large animal model of autoimmune diabetes to facilitate the translational research of autoimmune diabetes in human and provide a valuable model for transplantation immunity.

为更好了解自身免疫性糖尿病的发病机制，并制定有效治疗方法，适当的动物模型非常关键。NOD小鼠和BB大鼠已被用来进行自身免疫性糖尿病的研究，但由于与人类差别较大，啮齿类动物模型并不能完全模拟人类疾病，也不能提供充分证据以将研究结果转化到人。我们近期研究诱导的恒河猴糖尿病模型，显示出了自身免疫性糖尿病的特征。基于此，本课题拟采用抗CD25mA去除Treg联合链脲佐菌素的新方法，创新性建立恒河猴自身免疫性糖尿病模型。通过空腹和餐后血糖、血常规、血生化，静脉葡萄糖耐量等检测胰腺功能，判断是否为糖尿病；然后，检测自身反应抗体，活检取肝、肾、胰、脾和淋巴结进行病理学和免疫组化检测，流式细胞术及自体胰岛细胞移植等方法检测胰腺淋巴细胞浸润等，验证是否为自身免疫性糖尿病；检测抗体及细胞因子等表达情况并探寻其中机理；建立特定评价体系。为药物筛选、细胞移植治疗方案的建立及转换医学研究提供可靠的临床前动物模型。

恒河猴在亲源关系上与人类非常接近。为更好了解人类自身免疫性糖尿病的发生发展过程，研究疾病特点，建立临床前研究的自身免疫性1型糖尿病大动物模型非常关键。本研究采用抗CD25mA去除Treg联合多次小剂量注射链脲菌素（STZ）的方法建立恒河猴自身免疫性糖尿病模型。观察动物基本状况和代谢情况，定期采血检测血液常规及血生化，静脉葡萄糖耐量检测胰腺功能，ELISA等方法检测胰岛自身抗体反应，动物活检取胰腺、肝脏和肾脏，进行病理学检查和免疫组化检测。流式细胞术及自体胰岛细胞移植等方法检测胰腺淋巴细胞浸润等。诱导后，单纯小剂量多次STZ注射组与STZ联合抗CD25单克隆抗体组均出现特异性胰岛素抗体、胰岛细胞抗体，和胰腺明显的特异性胰岛细胞破坏及T淋巴细胞浸润。恒河猴糖尿病模型差别不大。STZ与Treg之间关系也未发现显著性差异。单纯小剂量多次STZ注射与STZ联合抗CD25mA均能够成功稳定诱导不可逆性的恒河猴糖尿病，发生发展的过程中，有针对胰岛细胞的自身免疫反应发生。