转人胰岛淀粉样多肽基因调控小型猪β细胞凋亡的研究

The islet amyloid polypeptide (IAPP) deposited in 95% of type 2 diabetes individual which caused the β-cell apoptosis by endoplasmic reticulum stress and lead to the insulin secretion inadequate. Except humans, non-human primate and cats, other species can't express an amyloidogenic toxic form of IAPP from this gene. The transgenic hIAPP rodent model of type 2 diabetes has been established, however the difference between the metabolism mechism of the glucose and lipid metabolism of human and that of rodent limited its application. Wether or not the minipig model established by hIAPP transgenesis will have highly similarity with the mechanism of human type 2 diabetes in β-cell apoptosis is still not understant. In this study, the promoter of insulin from porcine will be constructed to the vector with one copy or multiple copies connected by 2A of hIAPP gene to make the transgenic pig. High sucrose diet will induced the hIAPP gene overexpressing in the β-cell, the amyloid peptide whether or not able to deposit in β-cell and lead to β-cell apoptosis will be evaluted and multiple copies of hIAPP gene whether or not able to increase the islet amyloid polypeptide deposition and intensify the amount of β-cell apoptosis than that of single copy will be assessed. The insulin resistance of peripheral tissues liver, muscle and fat will be detected, then elaborate the β-cell apoptosis induced by hIAPP genes in pig and the characteristics of transgenic pig similarity with that of human in type 2 diabetes for establishing the β-cell apoptosis regulation model in miniature pig.

胰岛淀粉样多肽沉积在95%人类2型糖尿病患者中经内质网应激引起β细胞凋亡，导致胰岛素分泌不足和糖代谢紊乱。除人、非人灵长类和猫外，其它物种胰岛淀粉样多肽基因均不能致病。将人胰岛淀粉样多肽（hIAPP）基因转入啮齿类后可获得2型糖尿病模型，然而啮齿类糖脂代谢与人的差异，限制了模型的应用。将hIAPP基因转入小型猪能否获得与人类2型糖尿病形成机理更相似的β细胞凋亡模型仍不清楚。本研究首先将猪胰岛素启动子与hIAPP基因单拷贝或由2A连接的多拷贝序列构建到载体，制备转基因小型猪；通过高糖饲料诱导hIAPP基因在β细胞中过表达，评价β细胞中是否能出现胰岛淀粉样多肽沉积和β细胞凋亡；以及多拷贝hIAPP基因能否使胰岛淀粉样多肽沉积量增大及β细胞凋亡程度加剧；对肝脏、肌肉和脂肪等组织胰岛素抵抗程度进行评价，分析转hIAPP基因猪2型糖尿病发生特征与人的相似性，为建立调控β细胞凋亡的小型猪模型奠定基础。

本项目以猪胰岛β细胞特异性表达胰岛素基因启动子及肝脏特异性表达载脂蛋白E基因启动子分别连接hIAPP、CHOP、GIPRdn和11β-HSD1基因，创制了三种携带人胰岛淀粉样多肽基因转多基因小型猪及一种转多基因小鼠，获得了共转多基因遗传变化规律，系统评价了hIAPP对猪β细胞功能影响及高脂高能量饮食引起的小型猪胰腺、肝脏、肾脏、心脏和骨骼肌组织器官分子病理机制。发现遗传过程中共转多基因与转染家系F0代的拷贝数直接相关，受载体类型及共转基因数影响较小；转入基因能高效特异性的在胰岛和肝脏中表达；hIAPP对β细胞凋亡影响的过程缓慢，组织特异性表达GIPRdn-hIAPP基因胰岛表现出代偿性增殖肥大特征；高脂高能量饮食促进胰岛、肝脏、肾脏、心脏和骨骼肌脂沉积，氧化应激通过线粒体信号通路（Bcl/Bax）活化caspase-3基因，促进β细胞凋亡；体内能量供应方式从葡萄糖转变到酮体触发早期非酒精性脂肪性肝炎，lncRNA参与肝脏免疫炎性、趋化因子活性、细胞因子活动和G蛋白偶联受体结合过程；异位脂沉积致使心肌肥大、炎性反应增强等病变；慢性肾损伤逐渐加剧；异位沉积的脂肪组织产生过量11β-HSD1，增高局部皮质酮水平，促使MSTN上调表达，抑制骨骼肌生长。本项目研究结果显示，小型猪2型糖尿病发展进程中所表现出来的病理变化特征与人类患者高度相似，是较好的人类疾病动物模型。