长爪沙鼠遗传性糖尿病模型群体的培育及其相关机制研究

Spontaneously diabetic animal model is one of precious materials for studying the pathogenesis mechanism and remedy of diabetes mellitus. The number of present genetic animal models for diabetes is less than 10 in the world and is limited to rodent animals. Furthermore, these models are preserved by the breeder and are protected by intellectual property rights. There is no model of genetic diabetes mellitus bred by Chinese researchers yet. Mongolian gerbil is one of particular laboratory animal originated from China. None of genetic model of diabetes mellitus in Mongolian gerbil has been reported. During our previous research work in selectively breeding inbred gerbil strain, we observed occasionally that animals from one of the sub-strains tend to have high blood glucose. After three generations selectively breeding by mating the high blood glucose animal each other, we confirmed that this character is obviously hereditary. We hypothesize that the high blood glucose character of these gerbils is resulted from gene mutations. Based on these results, we will selectively breed a genetic diabetes model of Mongolian gerbil by selecting and mating high blood glucose and impaired glucose tolerance animals in each generation. We will also investigate its pathogenesis in morphology, pathology and biochemical parameter to learn the character of this model. The diabetes related genes will further be detected by RT-PCR, immune-histochemistry method and Western-blotting to learn the level of these genes'expression in different tissues and organs of the animal in different ages. These data will illustrate the molecule mechanism and heredity character of diabetic gerbil model. Our study will establish a new animal model of diabetes mellitus and will provide a new material for researches on diabetes mechanism, development and evaluation of new drug in remedy of diabetes. Our implement of this study will also provide creative methods and novel targets for diabetes. It will be a substantial foundation for further selecting the candidate genes of diabetes.

遗传性糖尿病动物模型是研究糖尿病发病机制和治疗药物的珍贵材料，目前世界上遗传性糖尿病动物模型不足10种，均局限于啮齿类，且由培育者所拥有，受产权保护。我国尚无自行培育的自发糖尿病动物模型。长爪沙鼠是我国特有实验动物，有关其自发糖尿病模型还未见报道。我们在长爪沙鼠近交系培育过程中偶然发现其中一个分支有自发高血糖趋势，经3代筛选高血糖动物定向培育，证明这种自发高血糖具有明显的遗传性，推测该分支动物发生了基因改变而出现高血糖。本研究以此为基础，采用每代选择高血糖和糖耐量异常的动物定向培育长爪沙鼠遗传性糖尿病模型群体。同时测试其生理生化、病理等生物学指标，阐明该模型发病特征。通过糖尿病相关基因在动物不同年龄不同组织中的表达分析，揭示该糖尿病模型发生的分子机制和遗传特性。本研究将建立一种新型的糖尿病动物模型，为糖尿病发病机制研究和药物开发提供新型材料，为下一步利用长爪沙鼠筛选糖尿病易感基因奠定基础。

糖尿病是全球关注的重大慢性疾病之一，也是主要影响中国人健康的疾病之一。遗传性糖尿病动物模型是研究糖尿病发病机制和治疗药物的珍贵材料，但世界上遗传性糖尿病动物模型不足10种，且局限于啮齿类。我国尚无自行培育的自发糖尿病动物模型。长爪沙鼠是我国特有实验动物，有关其自发糖尿病模型尚未见报道。本课题中，我们在前期培育长爪沙鼠近交系过程中偶然发现的一个分支的基础上，继续将该动物品系从第F3代定向培育到F10代（总代数17代），满足模型标准（空腹血糖（FPG）≥5.2且口服葡萄糖溶液后2h的血糖值(PG2h)≥6.8mmol/L）的动物比例从F0代的21.33%到F9/F10超过66%，建立了比较稳定的自发性糖尿病长爪沙鼠模型群体，留有73对种子动物。用28个微卫星位点对F0-F6进行遗传监测的结果表明，群体没有发生遗传污染。检测不同年龄动物的FPG和PG2h、FINS、FFA、TG、HDL-C、LDL-C、TC等生化指标，以及血清中LEP和ADP水平发现FPG、LEP和ADP等糖尿病指标在各年龄段均比较稳定，说明成年后该模型比较稳定。对F2-F7代166只动物的生化指标检测表明，各代动物的各项生化指标无显著差异。用光学/电子显微镜观察糖尿病长爪沙鼠的多种组织病理形态学结构发现，该模型动物的胰腺、肝脏和肾脏中均出现不同程度的病变。用RACE法克隆了5种糖尿病候选基因葡萄糖转运子4(GLUT4）、脂联素(Adiponectin，ADP)、瘦素（Leptin，LEP）、蛋白激酶B(Akt)、钙蛋白酶10（CAPN10）等，用SSH筛选到SLC39A7等26个与糖尿病相关的基因，并用免疫组化、Realtime PCR和Western blot检测了这些基因在动物各种组织中的含量和表达水平，发现这些基因在各组织器官中的表达水平变化各有特点，说明它们在长爪沙鼠糖尿病的发生中发挥不同作用，也说明该模型动物糖尿病的发生与遗传因素有关。本研究建立了一种新型的糖尿病动物模型，可以为糖尿病发病机制研究和药物开发提供一种新型材料，也为下一步利用长爪沙鼠筛选糖尿病易感基因奠定基础。