基于类胰岛素分泌缺陷的新型糖尿病动物模型的构建及其在降糖药物筛选中的初步应用

Diabetes is a complex and heterogeneous disorder presently affecting more than 100 million people worldwide and causing serious socio-economic problems. Appropriate experimental models are essential tools for testing of various therapeutic agents and understanding the pathogenesis, complications, and genetic or environmental influences that increase the risks of diabetes. Animal models of lacking insulin are used for study on pathogenesis of diabetes and hypoglycemic drug screening, however little research has been done on silkworm diabetic model , which has advantages over traditional animal diabetic models in high screening efficiency, low cost and simple manipulation. 32 bombyxin genes that exclusively expressed in four pairs of the medial neurosecretory cells in the brain (insulin-producing cells in Bombyx mori, BPCs) are in contrast to the insulin gene, expressed in the pancreatic β cell. Bombyxin comprises a mixture of highly heterogeneous molecular forms whose amino acid sequences have 40% identity with human insulin and has been considered as a critical factor regulating carbohydrate metabolism. To establish silkworm as a relevant model for metabolic disorders, the apoptotic gene ICE (Interlukin-1-β converting enzyme) will be driven by a QF transgene whose expression was determined by a fragment of the Bombyxin A1 promoter to express exclusively in the neurosecretory cells of larval brains. A major role of BPCs in carbohydrate metabolism will be validated by altered glycogen and triglyceride stores as well as increased circulating lipid levels measured in BPCs-ablated silkworms. Finally, We will examine whether it could be evaluated using BPCs-ablated silkworms for the hypoglycemic effect of anti-diabetic drugs as insulin, rosiglitazone, metformin and whether geniposide as the main active ingredient of traditional chinese medicine gardenia could decrease the hemolymph sugar levels of the hyperglycemic silkworms. Our project will provide experimental evidence for research and development and application of silkworm diabetic model.

糖尿病动物模型在其发病机制的研究和降糖药物的筛选及药效评价中具有重要的作用，基于经济、高效的无脊椎动物——家蚕研发的类胰岛素(家蚕素, Bombyxin，BBX)缺失的糖尿病动物模型尚未见报道。家蚕脑中央背部的4对神经分泌细胞(BPC)合成参与糖代谢调节类胰岛素——家蚕素基因，与哺乳动物胰岛β细胞功能相似。本研究拟探索基于特异表达在BPC神经细胞中的BBX-A1启动子驱动表达家蚕凋亡基因BmICE，采用可抑制新型双元表达Q系统，通过转基因家蚕杂交获得类胰岛素分泌缺陷型家蚕，在个体水平上检测并评价类胰岛素缺失对家蚕糖代谢的影响；最后，类胰岛素分泌缺陷型家蚕通过注射胰岛素及人工添食罗格列酮、二甲双胍、京尼平苷等药物，探索其作为降糖药物筛选模型的可行性，并评价中药栀子的主要活性成分京尼平苷的降糖效果。本项目旨在为基于家蚕的糖尿病模型的研发及应用提供依据。

糖尿病已成为继心血管疾病、肿瘤之后第三位危害人类健康的慢性疾病。由于糖尿病的病因不明，诱发因素较多，糖尿病动物模型作为人类糖尿病病理生理的最直观体现，至今仍是医学研究糖尿病发病机制、筛选糖尿病活性药物最常用、最直观的研究工具。目前小鼠和大鼠是最常用的糖尿病研究及药物筛选的动物模型，但是大规模药物初筛需要大量的动物，存在高成本、与伦理冲突等问题，所以使用无脊椎动物用于糖尿病研究及药物筛选为越来越多研究者所接受。家蚕理论上实用于人类糖尿病的发病机制和开发新降糖药物的研究，但相关研究报道很少。. 本研究用不同浓度高糖饲料家蚕后，导致血液中葡萄糖和海藻糖含量显著升高；脂肪组织中甘油三酯积累，脂肪球明显增大、增多；家蚕体重明显降低，生长异常；人胰岛素对高糖饲料饲养的家蚕血液中葡萄糖有显著降低作用。通过蛋白质组学研究技术探索其代谢异常的发生机制，表明家蚕中血糖升高可能部分是由于葡萄糖转运体4（GLUT4）定位于细胞膜受阻，从而阻碍葡萄糖的摄取和利用。. 通过过量表达BmFoxO的转基因家蚕（Over-FoxO）获得家蚕糖尿病模型：Over-FoxO家蚕的幼虫、蛹和蛾个体明显变小，体重显著降低，血淋巴中葡萄糖和海藻糖含量显著增加，而甘油三酯含量降低，脂肪体中糖原含量降低。通过定量分析和代谢组学技术表明过表达BmFoxO后抑制了家蚕体内蛋白质和核糖体的合成使家蚕生长受到抑制，并且促进了家蚕体内的糖异生和脂类分解。. 本研究结果表明家蚕在高糖喂养具有与人类糖尿病相似的症状，说明家蚕有望作为一种新型的无脊椎动物模型应用于饮食导致的糖尿病发病机制及其用家蚕用于药物初筛的研究。