定点共转多基因构建小型猪2型糖尿病模型

Type 2 diabetes mellitus is seriously endangered human health. There is significant difference between the rodent disease models of type 2 diabetes and human patient in the adipose tissue characteristics, inflammatory mechanism, lipid profile and intestinal flora etc. which closely related to the type 2 diabetes mellitus development. Available minipig models of type 2 diabetes, induced by high-fat and high caloric diet or chemical drugs or single gene modified, exhibit similar pathological characteristics of human patient in stage. However, there is no stable genetic minipig model of type 2 diabetes performed the completely pathological characteristics in disease development and complications. The multi-transgenic and site-specific integration technology will be carried out by this project to knockin three genes of human PNPLA3I148M polymorphism, GIPRdn mutation and hIAPP in the H11 loci of minipig together, realize the tissue specific expression of these genes in liver and pancreatic islet β-cell respectively, for constructing the type 2 diabetes model of minipig with the characteristics of liver lipid deposition inducing insulin resistance and β cell proliferation inhibition companied with apoptosis increasing. According to the development process of type 2 diabetes mellitus, the evaluation of clinical pathology of minipigs induced by high-fat high-energy diet and normal diet will conducted from three stages and nine categories. At last, the core breeding herd of type 2 diabetes model of minipig will be established. With completion of this project, the type 2 diabetes model of minipig with highly similar pathogenesis followed human will provided for the research and application in pathological mechanism studying, drug target screening and drug safety evaluation.

2型糖尿病严重危害人类健康。啮齿类2型糖尿病模型在疾病发生密切相关的脂肪组织特性、炎性机制、脂蛋白谱和肠道菌群结构等方面与人类存在显著差异。高脂高能量饮食、化学药物诱导或单基因修饰可获得具有典型2型糖尿病阶段性病理特征的小型猪模型，但至今尚无遗传稳定，完整展现2型糖尿病发生发展及并发症病理进程的小型猪模型。本项目通过多基因定点整合技术，将人PNPLA3I148M多态、GIPRdn突变体和hIAPP三基因共转到猪H11位点，分别在肝脏和胰岛β细胞组织特异性表达目的基因，创制肝脏脂沉积促发胰岛素抵抗，胰岛β细胞增殖受抑制伴随凋亡增加的小型猪2型糖尿病模型。根据2型糖尿病发生发展过程，从三个阶段、九个层面对高脂高能量饮食和普通饮食诱导的小型猪进行临床病理学评价，组建小型猪2型糖尿病核心群。本项目完成后，将为2型糖尿病发生机理、药物靶标筛选和新药安全评价等方面研究提供病理机制高度相似的小型猪模型。

2型糖尿病严重危害人类健康，高脂高能量饮食、化学药物诱导或单基因修饰可获得具有阶段性病理特征的小型猪2型糖尿病模型。在该项目实施前尚无遗传稳定，展现2型糖尿病多阶段病理特征的小型猪模型。该项目通过多基因定点整合技术，将3个人类代谢性疾病易感基因（GIPRdn、hIAPP和PNPLA3I148M）转入猪基因组，经组织特异性启动子调控GIPRdn和hIAPP在胰岛β细胞中的表达，调控人PNPLA3I148M在肝脏中的表达。经基础日粮、高糖饲料和高脂高能量饲料三种饮食方式，进行了多种模型构建方案的评估，获得2型糖尿病及并发症相关组织器官，胰腺、肝脏、脂肪、骨骼肌、心脏和肾脏等的组织病理学特征。结果显示，经13个月的基础日粮饲喂，转基因猪表现出皮下脂肪减少、胰岛素抵抗、胰腺脂沉积和肝脏炎症逐渐增加等特征；5月龄转基因猪经1个月高糖饮食，空腹血糖上升到7.0以上；9月龄转基因猪经高脂高能量饲喂3个月后，转基因猪表现出葡萄糖耐量受损、脂肪性胰腺、脂肪和肝脏慢性炎症等病理特征，饲喂时间延长至5个月和18个月，胰腺纤维化和钙化特征逐渐加重，肝脏脂沉积和纤维化逐渐加重、出现大量假小叶，心肌线粒体受损、细胞肿胀，肾小球从基底膜增厚发展到肾小球萎缩。针对高脂高能量饮食初期，转基因猪糖脂代谢指标改善的情况，通过脂肪和骨骼肌组织转录组，结合病理组织学等研究，阐述了肠系膜脂肪及骨骼肌在机体对富营养和体重增加产生适应性变化的同时触发代谢紊乱的特征。针对转基因猪短期高脂高能量饮食表现出典型的炎症特征，详细解析了转基因猪模型肝脏和脂肪组织CD8+T细胞触发代谢炎症级联反应的分子特征，以及炎症发生过程中关键基因在物种间的相似性。该研究获得的转三基因猪模型表现出2型糖尿病发生过程中胰腺、肝脏、脂肪、心脏和肾脏等组织器官典型的分子病理特征，可应用于这些组织器官分子病理机制研究，以及药物靶点开发和转化医学评价研究。

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