【作者】 魏杰；

【导师】 徐顺清；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生， 2011， 博士

【摘要】 2型糖尿病的发病率在全球范围内日益增加,现已成为21世纪危害人体健康的主要疾病之一。胰岛素抵抗和进行性的β细胞功能损害是2型糖尿病的两个重要特征；其中胰岛β细胞功能损害是糖尿病发生、发展的必要条件。传统观点认为,遗传、无节制的高热量饮食和久坐不动的生活方式的改变是导致2型糖尿病发生发展的主要因素。但是,目前这些因素已经不能完全解释2型糖尿病发病率持续增加的现象。最近,越来越多的研究表明,日常生活中主动或被动接触的外源化合物也可以干扰胰腺β细胞的功能,从而在2型糖尿病发生的过程中发挥着举足轻重的作用。邻苯二甲酸二(2-乙基己基)酯[Di-(2-ethylhexyl)-phthalate,DEHP]是生产聚氯乙烯(PVC)的一种主要增塑剂,在日常生活中广泛应用。DEHP可通过消化系统、呼吸系统及皮肤接触等途径进入人体,也可通过胎盘和乳汁到达下一代体内。在美国进行的一项流行病学调查发现,75%的被调查者尿液中可以被检测到DEHP及其代谢物。目前已有部分流行病学和动物实验显示,DEHP暴露和机体血糖代谢紊乱存在一定的联系。由于胎儿和新生儿对环境污染物非常敏感,所以本研究建立了发育关键时期DEHP暴露的实验动物模型,探讨围产期DEHP暴露对子代机体糖代谢的影响,并从胰岛β细胞的发育、胰岛素分泌功能以及凋亡等方面探讨其可能的机制。第一部分围产期DEHP暴露对子代大鼠糖代谢的影响目的：探讨围产期DEHP暴露对子代大鼠生长发育和糖代谢的影响。方法：Wistar雌鼠阴道涂片检查确定受孕,从受孕0天开始进行玉米油和不同剂量的DEHP (1.25和6.25 mg/kg/day)灌胃染毒直至仔鼠断乳(出生后第21天)。测量孕鼠孕期和泌乳期的体重以及断乳时的血糖、胰岛素水平的变化。断乳后每组保留雌雄仔鼠各16只进行后续的实验。动态监测子代生长过程中体重、摄食量、血糖、胰岛素、葡萄糖耐量和胰岛素耐量的变化。同时分析统计子代断乳时和成年后性腺旁脂肪组织的重量,并观察脂肪细胞的形态。结果：与对照组相比,围产期DEHP暴露引起子代出生体重显著减小；出生后生长速度明显减慢。断乳时,DEHP暴露组子代出现体脂系数和脂肪细胞面积显著减小；空腹血糖和空腹胰岛素显著降低。成年后,DEHP暴露组子代雌鼠出现严重的葡萄糖代谢紊乱,主要表现为高血糖、低胰岛素血症和葡萄糖严重耐受不良；雄鼠空腹血糖与对照相比无明显差异,但空腹胰岛素显著升高。围产期DEHP暴露未改变孕鼠的体重、血糖和胰岛素水平。结论：围产期DEHP暴露引起子代大鼠生长发育速度减慢和机体糖代谢紊乱。第二部分围产期DEHP暴露对子代大鼠内分泌胰腺发育的影响目的：通过围产期DEHP暴露引起子代大鼠糖代谢紊乱的动物模型,探讨DEHP暴露对子代大鼠内分泌胰腺的早期影响。方法：用阴道涂片法检测受孕情况,从Wistar大鼠雌性受孕后0天(GD0)开始,对其进行玉米油和不同剂量DEHP (1.25和6.25mg/kg/day)灌胃染毒直至仔鼠出生后第21天断乳。于PND21处死部分仔鼠,收集胰腺样本、称重并测量胰腺组织中胰岛素的含量。免疫组化分析β细胞形态和质量；电镜观察β细胞的超微结构。QPCR分析仔鼠胰腺组织中Pdx-1、insulin、glucagon、Atf4、Atf6、Bip、Ucp2的mRNA表达水平；Western Blot分析PDX-1和磷酸化的PERK蛋白含量。结果：与对照组相比,实验组子代断乳时β细胞质量和胰腺胰岛素含量显著降低。电镜结果提示围产期DEHP暴露导致断乳时子代大鼠β细胞超微结构损伤,主要表现为：内质网扩张脱颗粒、线粒体肿胀、完整的分泌颗粒减少、不成熟的分泌颗粒增加。围产期DEHP暴露还导致子代断乳时胰腺组织中Pdx-1、insulin的表达显著下降,以及Atf4、ATF6、BiP、Ucp2 mRNA的表达水平明显增加；glucagon的表达水平未受影响。蛋白表达水平与mRNA的表达水平意义一致。结论：围产期DEHP暴露导致早期内分泌胰腺发育受损,进而引起子代大鼠成年后出现的葡萄糖代谢紊乱。第三部分围产期DEHP暴露导致子代大鼠成年后p细胞的功能损伤目的：通过围产期DEHP暴露引起子代大鼠糖代谢紊乱的动物模型,探讨DEHP暴露对子代成年大鼠内分泌胰腺形态和功能的影响。方法：将健康受孕Wistar大鼠随机分为对照组(玉米油2 ml/kg/day)、低剂量组(DEHP 1.25 mg/kg/day)和高剂量组(DEHP 6.25 mg/kg/day).各实验组从受孕0天开始至产后第21天断乳(GDO～PND21)持续灌胃染毒。子代饲养至出生后第27周,收集胰腺组织,称重,测量胰岛素含量。电镜观察β细胞的超微结构；免疫组化分析β细胞面积和质量。利用葡萄糖刺激的胰岛素分泌实验分析离体培养β细胞的分泌功能。Western Blot分析胰腺组织中Bax、Bcl-2、Fas、FasL以及casepase-3细胞凋亡相关蛋白表达水平。结果：围产期DEHP暴露导致子代雌鼠成年后胰腺β细胞面积、β细胞质量以及胰腺胰岛素含量明显低于对照；离体培养的胰岛细胞分泌功能也显著降低。DEHP组雄鼠β细胞的面积和质量与对照相比,无统计学差异。但DEHP组雄鼠单位胰腺胰岛素的含量和低糖刺激下胰岛细胞分泌胰岛素的能力显著高于相应的对照组。电镜结果证实DEHP组β细胞超微结构的损伤随着年龄的增加,损伤程度不断加重。值得注意的是,DEHP暴露还导致了子代雌鼠成年后胰腺中凋亡相关蛋白,如Bax/Bcl-2、Fas、FasL、casepase-3表达水平显著升高。结论：围产期DEHP暴露导致子代成年大鼠的葡萄糖代谢紊乱与成年内分泌胰腺的凋亡密切相关。更多还原

【Abstract】 The incidence of type 2 diabetes is dramatically increasing at an alarming rate worldwide and is regearded as one of the main threats to human health in the 21 St century. Reduced insulin sensitivity andβcells dysfunction are the core pathophysiological defects in type 2 diabetes. It has recently been acknowledged that (3 cells failture is the triggering factor for the progression of diabetes. Accepted risk factors such as dietary, lifestyle and genetics cannot fully explain the prevalence of diabetes mellitus, and there is increasing evidences suggesting that the increased presence of environmental pollutants plays an important role in the elevated incidence of type 2 diabetesDi(2-ethylhexyl) Phthalate (DEHP), one of the most widespread plasticizer to impart flexibility of polyvinyl-chloride (PVC), is commonly used in the manufacture of many dairy products. General human population is widely exposed to DEHP in everyday life through inhalation, ingestion or skin contact. It has been also proven that DEHP can easily cross the placenta and enter breast milk, and then is rapidly absorbed by offspring. A recent epidemiological study indicates that more than 75% of the U.S. population has been detected measurable levels of DEHP and other phthalate metabolites in the urine. Recently, mounting evidence has also point to the impact of DEHP exposure on metabolic state. As it is generally accepted that individuals are more sensitive to chemical exposure during vital developmental periods; in the present study, we used an animal model to investigate whether developmental DEHP exposure disrupts glucose homeostasis in the rat and whether this was associated with the early impairment in developing endocrine pancreas. We also examined the direct effect of developmental DEHP exposure on insulin secretion and apoptosis ofβcellsPart I Effects of Perinatal DEHP Exposure on Glucose Metabolism in the Rat OffspringObjective:To study the impact of perinatal DEHP exposure on the growth and the whole-body glucose homeostasis in rat offspring. Methods:Pregnant Wistar rats were administered corn oil,1.25 and 6.25 mg/kg/day DEHP by gavage from gestation day 0 (GDO) to weaning at postnatal day 21 (PND 21). Body weight of dams was measured during gestation and lactation. Fasting glucose and insulin in dams were determined on PND21. Body weight, food intake, blood glucose, serum insulin, glucose and insulin tolerance test in offspring were measured during the growth. In addition, perigonadal adipose tissues mass, body fat percentage and area of adipocyte were measured at weaning. Results:Relative to controls, DEHP-exposed offspring had a significantly lower birth weight and maintained relatively lower body weight up to 27 weeks of age. At weaning, glucose-lowering effects were much more pronounced in DEHP-exposed offspring of both sexes, which accompanied by the decreased adipocyte size and body fat percentage. At adulthood, female DEHP-exposed offspring exhibited elevated blood glucose, reduced serum insulin and impaired glucose tolerance. Male DEHP-exposed offspring had increased serum insulin, although there were no significant differences in blood glucose at fasting and during glucose tolerance test. Body weight, fasting blood glucose and serum insulin were comparable between control and DEHP-exposed dams. Conclusions:Perinatal exposure to DEHP gives rise to growth retardation and the whole-body glucometabolic abnormalities in the rat offspring. PartⅡEffects of Perinatal DEHP Exposure on the Development of Endocrine Pancreas in the Rat OffspringObjective:In this study, we investigated the impact of perinatal DEHP exposure on the early endocrine pancreas development in rat pups before and at weaning. Methods: Pregnant Wistar rats were administered DEHP (1.25 and 6.25 mg/kg/day) or corn oil throughout gestation and lactation by oral gavage. Pancreas weight and pancreatic insulin content were measure at weaning (PND 21).βcells mass, area and morphology were determined by immunofluorescence.βcells ultrastructure was measured by transmission electron microcopy. In addition, mRNA levels of Pdx-1、insulin、glucagon、Atf4、Atf6、Bip and Ucp2 and protein levels of PDX-1 and p-PERK in pancretic tissue were determined at weaning. Results:DEHP exposed offspring had lower pancreas weight and pancreatic insulin content than controls at weaning. Abnormalβcells untrastructure, characterized as hypertrophic rough endoplasmic reticulum, swollen mitochondria, increased proportion of immature and reduced proportion of filled granule, was observed in DEHP-exposed offspring at this time point. All these alterations induced by DEHP corroborates with changes in genes expression in pancreas, such as Pdx-1, insulin, glucagon, Atf4, Atf6, Bip and Ucp2. Moreover, decreased potein levels of PDX-1 and increased potein levels p-PERK were observed in DEHP-exposed offspring. Conclusion:Perinatal exposure to DEHP impairs endocrine pancreas early in life and leads to severe glucometabolic disorders in adult rat offspring. PartⅢPerinatal DEHP Exposure in Wistar Rats Offspring Causesβcells Dysfunction at AdulthoodObjective:In this study, we examined effects of fetal and neonatal exposure to DEHP onβcells structure and function in rat offspring at adulthood. Methods:Pregnant Wistar rats were given corn oil or DEHP (1.25 and 6.25mg/kg/day) via gavage from GD 0 until weaning at PND 21. Offspring were fed a normal diet after weaning and were raised from PND 21 to the age of 27 week. At week 27, pancreas tissue was collected for immunohistochemistry and transmission electron microscopy. Glucose-stimulated insulin secretion ex vivo was measured to evaluateβcells function. Key markers of each apoptotic pathway, such as Bax、Bcl-2、Fas、FasL and casepase-3 were examined in whole pancreas homogenates by Western blotting in female offspring. Results:At week 27, decreased pancreatic insulin content, reduced beta cells mass and area and evident degranulation of beta cells was observed in female DEHP-exposed offspring compared with controls. Diminished ability to secrete insulin in response to Glucose stimulus ex vivo was also observed following developmental DEHP exposure. In males, there was no difference in beta cells mass and area between DEHP treated offspring and controls, but elevated pancreatic insulin content and insulin secretion ex vivo was observed in DEHP treated offspring. Observed defects in P ultrastructure progressively got worse with age, even though DEHP exposure was discontinued at weaning. Importantly, Bax/Bcl-2, Fas, FasL and casepase-3 protein were significantly increased in the DEHP exposed female offspring compared with controls. Conclusion:Perinatal DEHP exposure predisposes offspring to glucose metabolic dysfunction at adulthood, and which was associated withβcells dysfunction and apoptosis.更多还原