版纳微型猪糖尿病模型及其Musclin的表达研究

发布时间：2018-04-22 02:00

本文选题：糖尿病 + 版纳微型猪　；参考：《南华大学》2012年硕士论文

【摘要】：背景及目的：糖尿病(DM)是一种严重危害人体健康的常见内分泌代谢疾病，DM的发生由胰岛素分泌缺乏及(或)其生物作用障碍引起，主要特征表现为高血糖。根据胰岛素缺乏的类型，可将糖尿病分为1型糖尿病和2型糖尿病，胰岛素抵抗(IR)是二者区分的关键。在DM发生过程中，有多种蛋白和细胞因子同时参与，共同调节着糖类和脂质代谢过程，阐明它们在DM中的作用机制，有利于积极预防和治疗DM。已有研究表明肌肉素(Musclin)能明显地抑制由胰岛素促进的葡萄糖摄入及糖元合成过程，推测Musclin与血糖、胰岛素之间存在某种联系，Musclin在DM的发生发展中起着重要作用，但具体作用机理仍不明确。因此，本论文在高糖高脂饮食喂养联合多次小剂量STZ注射成功建立版纳微型猪糖尿病模型的基础上，观察Musclin在版纳微型猪血清及各组织中的表达情况，并初步探讨肌肉素(Musclin)在糖尿病中的作用。方法：1.实验动物及分组：8头版纳微型猪，全雄性，2月龄，随机分为两组：正常对照组(CD)；高糖高脂+STZ注射组(HFSD+STZ)。2.糖尿病模型的建立：CD组饲以基础饲料；HFSD+STZ组饲以高糖高脂饲料，在第1个月的第1周按50mg/kg体重腹腔注射1%的STZ，在饲养第7个月的第1周连续3次通过耳缘静脉注射STZ (每天1次)，每次注射量为50mg/kg体重。CD组版纳微型猪仅注射等体积的柠檬酸一柠檬酸钠缓冲液。建模期12个月。3.血清生化指标的检测：每个月末采用葡萄糖氧化酶-过氧化物酶法检测空腹血清葡萄糖浓度、直接化学发光法检测胰岛素浓度、甘油磷酸氧化酶-过氧化物酶法检测甘油三酯和胆固醇氧化酶-过氧化物酶法检测总胆固醇浓度，并在第12个月末进行静脉葡萄糖耐量试验(IVGTT)和改良静脉糖耐量试验；每个月末采用ELISA法检测血清中的Musclin含量。4.第12个月末处死动物，，分离心肌、肝脏、肾脏、骨骼肌和胰腺组织，HE染色，光镜下观察各组织的形态结构，电镜下观察各组织的超微结构，PAS染色观察肌糖原及肝糖原合成情况,苏丹IV染色观察脂质沉积情况。5.Western Blot方法及免疫组织化学法分别检测Musclin在版纳微型猪骨骼肌、肝脏、肾脏和胰腺中的蛋白表达和蛋白定位情况。结果：高糖高脂饮食喂养联合多次小剂量STZ注射导致版纳微型猪的糖代谢和脂代谢发生紊乱：血糖升高，血清胰岛素浓度下降，糖耐量减退及胰岛素敏感性降低，同时出现血清甘油三酯、总胆固醇浓度明显升高。在光镜、电镜下，各组织结构发生了不同程度的病理改变，肌糖原和肝糖原的合成减少，肝脏和胰腺组织内出现脂质蓄积。高糖高脂饮食喂养加STZ注射后，版纳微型猪血清中Musclin的含量增加，到第5个月增加显著，6月Musclin表达达到高峰，7月开始下降，但仍高于CD组。HFSD+STZ组版纳微型猪肝、肾、胰腺和骨骼肌组织中Musclin蛋白的表达明显升高，与CD组相比差异显著。结论： 1.高糖高脂饮食联合STZ注射可诱导版纳微型猪出现高血糖、低胰岛素血症、高甘油三酯血症和高胆固醇血症，发生糖耐量减低，胰岛素敏感性降低，组织结构出现病理改变，成功建立版纳微型猪糖尿病模型。 2. HFSD+STZ注射可上调版纳微型猪血清中Musclin的表达及肝、肾、骨骼肌和胰腺组织Musclin蛋白的表达。推测Musclin参与糖代谢过程，可能诱发胰岛素抵抗。
[Abstract]:Background and objective: diabetes (DM) is a common endocrine and metabolic disease that seriously endangering human health. The occurrence of DM is caused by the lack of insulin secretion and (or) its biological disturbance. The main characteristics are hyperglycemia. According to the type of insulin deficiency, diabetes can be divided into type 1 diabetes and type 2 diabetes, insulin resistance (IR). It is the key to distinguish between the two. In the process of DM, a variety of proteins and cytokines are involved in the simultaneous regulation of carbohydrate and lipid metabolic processes, and the mechanism of their action in DM is clarified, which is beneficial to the active prevention and treatment of DM., which indicates that the muscle element (Musclin) Neng Ming significantly inhibits the glucose intake and sugar promoted by insulin. In the process of meta synthesis, we speculate that there is a connection between Musclin and blood glucose and insulin, and Musclin plays an important role in the development of DM, but the specific mechanism is still not clear. Therefore, on the basis of the successful establishment of a miniature swine diabetes model with high glucose and high fat diet feeding combined with multiple small dose STZ injection, this paper observed Musclin In Banna miniature pig serum and tissues, the expression of myostatin (Musclin) in diabetes mellitus was preliminarily investigated.
Methods: 1. experimental animals and groups: 8 Banna miniature pigs, full male, 2 month old, were randomly divided into two groups: normal control group (CD), high glucose and high fat +STZ injection group (HFSD+STZ).2. diabetes model: the CD group was fed with basal diet, the HFSD+STZ group was fed with high glucose and high fat feed, and 1% was injected into the abdominal cavity by 50mg/kg body weight at first weeks of first months. STZ, STZ (1 times per day) was injected through the auricular vein 3 times in the first week of seventh months of feeding, each injection of 50mg/kg body weight.CD Banna mini pig was only injected with equal volume of citrate sodium citrate buffer. The serum biochemical indexes of.3. in the modeling period of 12 months were detected by the glucose oxidase peroxidase method at the end of each month. Serum glucose concentration was measured on the fasting serum, the concentration of insulin was detected by direct chemiluminescence, glyceric phosphate oxidase peroxidase assay was used to detect triglyceride and cholesterol oxidase peroxidase method to detect total cholesterol concentration, and the intravenous glucose tolerance test (IVGTT) and improved intravenous glucose tolerance test were carried out at the end of twelfth months. The Musclin content in serum was detected by ELISA method at the end of twelfth months, and the animals were killed at the end of twelfth months. The myocardium, liver, kidney, skeletal muscle and pancreas tissue were separated. The morphology and structure of the tissues were observed under the light microscope. The ultrastructure of the tissues was observed under the light microscope. The synthesis of glycogen and liver glycogen was observed by PAS staining. The lipid precipitation was observed by IV staining in Sultan. The lipid precipitation was observed by IV staining. The protein expression and protein localization of Musclin in the skeletal muscle, liver, kidney and pancreas of Banna miniature pig were detected by.5.Western Blot method and immunohistochemistry.
Results: high glucose and high fat diet combined with multiple small dose of STZ injection led to the disorder of sugar metabolism and lipid metabolism in Banna miniature pigs: blood glucose increased, serum insulin concentration decreased, glucose tolerance and insulin sensitivity decreased, serum triglyceride and total cholesterol concentration increased significantly. Under light microscope, electron microscope, each tissue The structure of the structure had varying degrees of pathological changes, the synthesis of muscle glycogen and liver glycogen decreased, lipid accumulation in the liver and pancreas tissue appeared. After high fat diet feeding and STZ injection, the content of Musclin in the serum of minitype pig increased significantly to fifth months, the expression of Musclin reached the peak in June and began to decline in July, but still higher than CD In group.HFSD+STZ, the expression of Musclin protein was significantly increased in Banna miniature pig liver, kidney, pancreas and skeletal muscle, and was significantly different from that in group CD.
Conclusion:
1. high glucose and high fat diet combined with STZ injection can induce hyperglycemia, hypoinsulinemia, hypertriglyceridemia, hypertriglyceridemia and hypercholesterolemia, lower glucose tolerance, lower insulin sensitivity and pathological changes in tissue structure.
2. HFSD+STZ injection can increase the expression of Musclin in the serum of minipig and the expression of Musclin protein in the liver, kidney, skeletal muscle and pancreas. It is presumed that Musclin participates in the process of glucose metabolism and may induce insulin resistance.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332

【参考文献】