壬基酚暴露与大鼠非酒精性脂肪肝发病关系及机制

发布时间：2018-06-05 03:25

本文选题：壬基酚 + 高脂高糖饮食　；参考：《遵义医学院》2017年硕士论文

【摘要】：非酒精性脂肪肝(NAFLD)在全球范围内广泛流行,发病率呈不断上升趋势,且病因不明。为探讨环境因素(壬基酚)和不良生活方式(高脂高糖饮食)在NAFLD发生发展过程中的作用,本课题分为两部分开展实验研究。第一部分,针对常规饲料喂养的SD大鼠,采用灌胃暴露壬基酚(NP)连续90天,研究NP在环境暴露浓度下是否可导致大鼠NAFLD发生;第二部分,针对高脂高糖饲料喂养的SD大鼠,采用NP连续90天灌胃,观察SD大鼠在高脂高糖饮食与NP共同作用下大鼠NAFLD发生情况,以及探讨NP亚慢性暴露是否加重或促进高脂高糖饮食所致NAFLD的发生。并检测与肝脏脂质合成相关基因、蛋白的表达,初步阐明NP致NAFLD的机制。第一部分NP亚慢性暴露对肝脏及功能的影响目的:探讨NP亚慢性暴露是否诱发大鼠非酒精性脂肪肝形成。方法:40只清洁级SD大鼠(200±20)g,雌雄各半,购于第三军医大学大坪实验动物中心(合格证号:(SCXK(渝)2012-0005))适应性喂养7天后随机分成4组,即对照组(玉米油),低剂量组(0.02μg/kg NP),中剂量组(0.2μg/kg NP),高剂量组(2μg/kg NP)。连续灌胃染毒90 d,灌胃期间每周称量体重1次,记录饮食量和饮水量。于第10周末每组大鼠随机选取4只进行肝脏超声检测,90 d后10%的水合氯醛麻醉后剖杀大鼠,腹主动脉取血分离血清用于肝功和血脂检测,称量肝脏质量并计算肝体指数,每只大鼠取肝脏最大叶边缘内侧1 mm处组织3等份,1份固定于10%甲醛用于HE染色、其余2份组织冻存-80度用于实时荧光定量PCR检测脂质代谢相关基因Fas,Srebp1,Ucp2的表达量,Western blot检测c/EBPα、PPARγ表达。结果:各NP暴露组体重,摄食和饮水无明显差异(P0.05)。超声结果显示,各组未见均未见明显脂肪肝改变。HE染色发现,仅高剂量NP组可见少量肝细胞脂肪变。肝功和血生化结果显示,血清ALT,AST,TC,LDL-C没有增加(P0.05)。RT-PCR结果显示,中,高剂量NP暴露组脂质合成相关基因Fas,Ucp2,Srebp1 m RNA与对照组表达均上调(P0.05)。Western blot结果显示,中、高剂量组脂质合成相关蛋白c/EBPα、PPARγ相对表达量明显上调(P0.05)。结论:低剂量NP亚慢性暴露未致大鼠非酒精性脂肪肝形成,但可上调部分肝脏脂质合成代谢相关基因Fas,Ucp2,Srebp1和蛋白c/EBPα、PPARγ的表达。第二部分NP联合高脂高糖饮食致大鼠非酒精性脂肪肝及机制研究目的:探讨NP亚慢性暴露是否加重高脂高糖饮食所致NAFLD的发生。方法:40只清洁级SD大鼠(200±20)g,雌雄各半,购于第三军医大学大坪实验动物中心(合格证号:(SCXK(渝)2012-0005))适应性喂养(100%基础饲料)7天后随机分成4组,即高脂高糖饮食对照组(玉米油),高脂高糖饮食+NP低剂量组(0.02μg/kg),高脂高糖饮食+NP中剂量组(0.2μg/kg),高脂高糖饮食+NP高剂量组(2μg/kg)。实验期间每周称重1次,记录饮水、摄食量。第10周末每组随机抽取4只大鼠进行肝脏彩色超声检查,出现强回声区可判断NAFLD模型建立成功。于第12周末对动物进行剖杀取材,10%的水合氯醛麻醉后,腹主动脉取血分离血清用于肝功和血脂检测。称量肝脏质量并计算肝体指数,每只大鼠取肝脏最大叶边缘内侧1 mm处组织3等份,1份固定于10%甲醛用于HE染色、其余2份组织冻存-80度用于实时荧光定量PCR检测脂质代谢相关基因Fas,Srebp1,Ucp2的表达量,Western blot检测c/EBPα、PPARγ表达。结果:高脂高糖饮食对照及NP暴露全部发生脂肪肝,第12周高剂量组体重增加(P0.05),低、中剂量NP暴露组饮水量,中、高剂量饮食量显著高于对照组(P0.05)。超声结果显示,各组均发现强回声区域,提示有脂肪肝形成,且中,高剂量组回声区强于对照组,高剂量组强于低剂量组。HE染色发现,NP暴露组随剂量增加大泡性脂肪变肝细胞逐渐增多。肝功和血生化结果显示,高剂量组ALT,AST,TC,LDL-C血清含量均增加,中剂量组TG,TC,LDL-C含量血清含量增加(P0.05)。RT-PCR结果显示,各NP暴露组Fas,Ucp2 m RNA表达量上调,中剂量Srebp1表达上调(P0.05)。Western blot结果显示,除低剂量NP暴露组PPARγ外,其余各组c/EBPα、PPARγ蛋白相对表达量均升高(P0.05)。结论:NP可加重高脂高糖饮食致大鼠非酒精性脂肪肝效应,其机制主要可能与上调Srebp1,Fas,Ucp2 m RNA表达,增强c/EBPα、PPARγ表达,增加TG合成有关。
[Abstract]:Non-alcoholic fatty liver (NAFLD) is widely prevalent throughout the world. The incidence of nonalcoholic fatty liver disease is increasing and the cause is unknown. In order to explore the role of environmental factors (Ren Jifen) and bad lifestyle (high fat and high sugar diet) in the development of NAFLD, this subject is divided into two parts. SD rats were used for 90 days of gastric exposure to nonylphenol (NP) for 90 days. The second part, for SD rats fed with high fat and high sugar feed, was administered by NP for 90 days to observe the occurrence of NAFLD in rats with high fat and high sugar diet and NP, and to explore NP sub. Whether chronic exposure aggravates or promotes the occurrence of NAFLD caused by high fat and high sugar diet, and detects the genes related to liver lipid synthesis, protein expression, and preliminarily clarifies the mechanism of NAFLD induced by NP. The first part of the effect of subchronic NP exposure on liver and function: To explore whether or not NP subchronic exposure induces nonalcoholic fatty liver formation in rats. Method: 40 clean SD rats (200 + 20) g and half male and half female were bought at the center of experimental animal of Daping experiment in Third Military Medical University (certificate number: (SCXK (Yu) 2012-0005) for 7 days. The control group was randomly divided into 4 groups, namely the control group (corn oil), the low dose group (0.02 g/kg NP), the medium dose group (0.2 mu g/kg NP), and the high dose group (2 micron NP). Continuous intragastric intragastric poisoning 90 d, The weight of diet and drinking water were recorded 1 times a week. At the end of the tenth week, 4 rats in each group were randomly selected for liver ultrasound examination. After 90 d, 10% of chloral chloral anaesthesia was killed in rats. The blood serum of abdominal aorta was used for liver function and blood lipid test, the liver quality was weighed and the liver body index was calculated. Each rat was taken the most liver. 3 equal parts were organized at the 1 mm of the medial margin of the large lobe, 1 parts were fixed to 10% formaldehyde for HE staining, and the other 2 tissues were frozen to be used for real-time fluorescence quantitative PCR to detect the expression of lipid metabolism related genes Fas, Srebp1, Ucp2, Western blot detection c/EBP a, PPAR gamma expression. The results showed that no significant fatty liver changes were found in each group, but only a small amount of hepatocyte fatty changes were found in the high dose NP group. The liver function and blood biochemical results showed that serum ALT, AST, TC, LDL-C did not increase (P0.05).RT-PCR results, in the high dose NP exposure group, the lipid synthesis related genes Fas, Ucp2, Srebp1 AST and control groups The P0.05.Western blot results showed that, in the high dose group, the lipid synthesis related protein c/EBP alpha and the relative expression of PPAR gamma increased significantly (P0.05). Conclusion: low dose NP subchronic exposure did not induce nonalcoholic fatty liver formation in rats, but could up regulate the liver lipid synthesis and metabolism related genes Fas, Ucp2, Srebp1 and protein c/EBP alpha, PPAR gamma Second part NP combined with high fat and high sugar diet induced nonalcoholic fatty liver and mechanism in rats. Objective: To explore whether NP subchronic exposure aggravates the occurrence of NAFLD caused by high fat and high sugar diet. Methods: 40 clean grade SD rats (200 + 20) g, and male and female, were purchased at the center of experimental animal in Daping of Third Military Medical University (certificate number: (SCXK) 2012-0005) after 7 days, adaptive feeding (100% base feed) was randomly divided into 4 groups, namely, high fat and high sugar diet control group (corn oil), high fat and high sugar diet +NP low dose group (0.02 mu g/kg), high fat and high sugar diet +NP dose group (0.2 mu g/kg), high fat and high sugar diet +NP high dose group (2 mu g/kg). During the experiment, weight 1 times a week, record drinking water intake, intake of food. At the end of the 10 week, 4 rats in each group were randomly selected for color ultrasonic examination of the liver, and the NAFLD model was established to determine the success of the hyperechoic region. At the end of the twelfth week, the animals were killed, and 10% of the hydrous chloral anaesthesia, the blood serum of the abdominal aorta was used to detect the liver function and blood lipid. 3 equal parts were organized at the 1 mm of the inner edge of the most lobe of the liver, 1 parts were fixed in 10% formaldehyde for HE staining, and the other 2 tissues were frozen to be used for real-time fluorescence quantitative PCR to detect the expression of lipid metabolism related genes Fas, Srebp1, Ucp2, Western blot detection c/EBP a, PPAR gamma expression. Fatty liver, Twelfth weeks high dose group weight increase (P0.05), low, medium dose NP exposure group drinking water volume, high dose diet was significantly higher than the control group (P0.05). Ultrasound results showed that all groups found the strong echo region, suggesting the formation of fatty liver, and the high dose group is stronger than the control group, the high dose group is stronger than the low dose group.HE staining found, NP The serum content of ALT, AST, TC and LDL-C increased in the high dose group, and the serum content of TG, TC and LDL-C in the middle dose group increased (P0.05).RT-PCR (P0.05).RT-PCR results in the middle dose group. The results of estern blot showed that the relative expression of c/EBP alpha and PPAR gamma in the other groups increased (P0.05) except the low dose NP exposure group PPAR gamma. Conclusion: NP can aggravate the nonalcoholic fatty liver effect in rats induced by high fat and high sugar diet. The mechanism is mainly related to the up regulation of Srebp1, Fas, Ucp2 m.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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