【作者】 刘芸；

【导师】 陈正礼；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2013， 硕士

【摘要】 本实验通过观察口服大豆异黄酮(SIF)对食源性肥胖(DIO)大鼠体重、腹部脂肪细胞形态以及中枢、胃肠道、心肺组织、肾脏中α-MSH蛋白和Ob-Rb mRNA、 POMC mRNA表达的影响,探讨SIF诱导Ob-Rb-POMC调控通路的可能,为SIF作用机制研究提供形态学依据。实验以4-5周龄体重140±10g的SPF级雄性大鼠(n=80)作为实验动物,10只作为基础对照组。其余大鼠采用高脂饲料喂养8周以建立食源性肥胖大鼠模型,后随机分4组,3组分别灌胃低(50mg/kg·BW)、中(150mg/kg·BW)、高(450mg/kg·BW)剂量SIF,剩余1组作肥胖对照组灌胃等量溶媒。基础对照组同期灌胃等量溶媒。所有大鼠每周称重。灌胃4周后,大鼠经4%多聚甲醛灌注固定并取腹部脂肪、大脑、垂体、空肠、胃、肺、肾、心脏行冰冻切片。运用苏丹Ⅲ染色观察腹部脂肪细胞形态,免疫组织化学SABC法检测各组织α-MSH蛋白表达,寡核苷酸探针原位组织杂交方法检测各组织POMC mRNA、Ob-Rb mRNA表达。实验结果显示：①DIO大鼠平均体重为363.91±2.46g(n=54),高于基础组(273.00±5.37g, n=12)+1.4个标准差,判断食源性肥胖大鼠模型建立成功；150、450mg/kg·BW SIF显著降低DIO大鼠体重；肥胖大鼠腹部脂肪细胞面积显著增大,分布不均、形态多样,150、450mg/kg·BW SIF显著减小腹部脂肪细胞面积,维持脂肪细胞分布和形态的均一性。SIY对肥胖具有下调作用。②DIO大鼠弓状核α-MSH蛋白、POMC mRNA、Ob-Rb mRNA表达下降,150和450mg/kg·BW SIF显著上调其表达；大鼠垂体表达α-MSH蛋白和少量POMC mRNA, SIF干预可增强其表达。③DIO大鼠肺α-MSH蛋白表达减少,Ob-Rb mRNA、POMC mRNA增多,450mg/kg-BW SIF可调整三者表达水平并使其接近于基础大鼠水平。④DIO大鼠胃底腺Ob-Rb mRNA表达下降,SIF显著增强其表达；仅在肥胖对照组大鼠胃底腺检测到POMC mRNA阳性信号。⑤空肠肠绒毛固有层均显示α-MSH蛋白、POMC mRNA阳性信号,且肥胖大鼠信号强度显著低于基础对照组。SIF灌胃显著提高其表达;Ob-Rb mRNA仅在中、高剂量组大鼠肠绒毛内表达。⑥大鼠肾仅检测到α-MSH蛋白阳性信号,定位于肾小球毛细血管和近曲、远曲小管；三者在DIO大鼠表达显著下降,可通过SIF干预逆转。⑦大鼠心肌细胞、微静脉和毛细血管表达α-MSH蛋白,其水平在DIO大鼠显著降低并可通过SIF干预逆转。结果显示,SIF干预可调节中枢和外周下丘脑、垂体、肺等组织α-MSH蛋白、POMC mRNA、Ob-Rb mRNA表达,从而改善肥胖大鼠中枢瘦素抵抗,缓解大鼠肥胖水平；并且α-MSH蛋白、POMC mRNA、Ob-Rb mRNA可能参与高脂高糖饮食诱导的胃粘膜损伤的形成,并与肥胖相关肺呼吸障碍和肾病有关,同时在维持机体正常血压方面可能具有一定作用。更多还原

【Abstract】 Objectives:To observe the role of oral SIF on DIO rats with respects to body weight and histologic morphology of abdominal adipocytes, detect expression levels of a-MSH protein,Ob-Rb mRNA, POMC mRNA in some tissues, discusse the possibility of Ob-Rb-POMC pathway induced by SIF, and provid morphology basis of the corresponding mechanisms.Methods:Eighty SPF sexually male rats weight140±10g aged4-5weeks old,feed with high fat and sugar diet for8weeks to copy DIO rats model, then randomly divided into four groups, respectively lavage with low (50mg/kg-BW), med (150mg/kg-BW), high (450mg/kg-BW) dose SIF and solven for4weeks, set standard control group (lavage with solvent) always. Measure body weights every week,then fixe the rats by perfusing4%paraformaldehyde and make frozen sections of abdominal fat, brain, pituitary gland, heart, lung, kidney, jejunum and stomach. Sudan Ⅲ dyeing is used to observe abdominal adipocyte, immunohistochemical SABC method to detect distribution and expression of a-MSH protein,oligonucleotide probe in situ hybridization method to distribution and expression of POMC mRNA and Ob-Rb mRNA.Achieved microscopic images and measured values of IOD.Results:(1) An average DIO rats weighed363.91±2.46g (n=54), higher than based control group (273.00+5.37g, n=12) plus1.4times of standard deviation,suggested succeed in copying DIO rats model;150and450mg/kg dose SIF significantly reduced DIO rats’weight; in DIO rats,abdomen adipocytes were significantly increased in area, various in morphological and distributed unevenly,150and450mg/kg SIF significantly decreased area of adipocytes,maintainedniformity of the distribution and morphological,shown a certain therapeutic effect on obesity.(2) Expression of a-MSH protein, POMC mRNA, Ob-Rb mRNA in ARC of DIO rats decreased,150and450mg/kg SIF significantly resisted the decline;a-MSH and a little POMC mRNA positive signal was detected in pituitary, SIF enhanced the expression.(3) Expression of a-MSH protein in lungs of DIO rats decreased,while Ob-Rb mRNA and POMC mRNA Increased,450mg/kg SIF regulated the expression to levels of based control rats.(4) Expression of Ob-Rb mRNA in gastric gland of DIO rats decreased, SIF significantly enhanced its expression; POMC mRNA positive signal was only detected in gastric gland of obese control rats.(5) Positive signals of a-MSH protein and POMC mRNA were detected in lamina propria of jejunum villi,which were weaker of obese rats than based control rats. SIF significantly increased the expression; Ob-Rb mRNA positive singal was only detected in jejunum villi of med and high dose groups.(6)Only a-MSH positive signal was detected in kidneys, specifically located at the glomerular,proximal and distal convoluted tubule, and decreased in obese rats,reversed by oral administration of SIF.(7) a-MSH positive signal was detected in myocardial cell nuclei and cardiovascular, its level decreased in obese rats, reversed by oral administration of SIF. Conclusions:Oral SIF regulated expression expression of a-MSH protein, POMC mRNA and Ob-Rb mRNA expression, thereby improving leptin resistance of DIO rats, down-regulated the level of obesity. And a-MSH protein and POMC mRNA, Ob-Rb mRNA may participate in Initiation of high-fat and high-sugar diet-induced gastric mucosal injury, obesity-related respiratory dysfunction and kidney disease, simultaneously play a role in maintain normal blood pressure and so on.更多还原