基于Tsc1甲基化调控mTORC1及下游关键因子的电针治疗单纯性肥胖大鼠机制研究

发布时间：2018-05-26 06:26

  本文选题：电针 + 单纯性肥胖　； 参考：《成都中医药大学》2016年硕士论文

【摘要】：目的：本研究以食源性肥胖(diet-induced obesity. DIO)大鼠模型为研究载体,从单纯性肥胖(simple obesity)发病的关键环节-下丘脑Tsc1-mTOR信号通路入手,在前期本研究组证实DIO大鼠下丘脑结节性硬化症因子1 (Tuberous sclerosis complex 1,Tscl)基因甲基化增加的基础上,观察下丘脑DNA甲基化转移酶(DNAMthyl-Transferases, Dnmts)对Tsc1基因的甲基化修饰作用,调控下游哺乳动物雷帕霉素靶蛋白复合物1 (mammalian target of rapamycin complex 1, mTORC 1)及关键因子在单纯性肥胖发病中的机制,从表观遗传学的角度观察针刺治疗肥胖的效应及机制。方法：选用刚断乳的健康清洁级,4周龄(体重70-90g)雄性SD(Sprague-Dawley)大鼠,剔除体重不符合要求的大鼠,然后随机分为造模组和普通饲料组。造模组运用改良后的高脂饲料喂养诱导DIO大鼠模型,造模14周后,将造模成功的DIO大鼠随机分为肥胖组(即模型组)和针刺组；普通饲料组用普通饲料喂养14周,剔除体重不符合要求的大鼠后作为对照组。针刺治疗：选取“天枢、中脘、三阴交和后三里”穴,用自制大鼠固定器固定大鼠后进行电针治疗,选疏密波,2/15Hz,30min/天,5天／疗程,休息2天,共4个疗程,对照组和肥胖组同法固定但不针刺。针刺治疗结束后称量体重并予以禁食12h,于次日取动物标本,用免疫组化标记法检测大鼠下丘脑弓状核Dnmts (Dnm2、Dnmt3a、Dnmt3b)的表达情况；用Western Blot蛋白质印迹法检测大鼠下丘脑弓状核mTORC1基因的蛋白表达情况；运用RT-PCR对大鼠下丘脑弓状核mTORC1及食欲肽(AgRP、NPY和PoMC) mRNA的含量及Tscl基因甲基化进行检测,分别比较各组结果差异。结果：1、体重结果：针刺干预前,肥胖组和针刺组DIO大鼠体重明显高于对照组(P0.01),肥胖组和针刺组组间体重无统计学差异(P0.05)；针刺干预治疗后,肥胖组和针刺组体重、体脂及体脂率均明显降低(P0.01)。2、下丘脑Tscl甲基化结果：(1)Tsc1片段1甲基化：与对照组相比较,肥胖组大鼠Tscl甲基化表达升高(P0.05)：电针干预治疗后,针刺组大鼠Tsc1甲基化表达降低,低于肥胖组(P0.05),针刺组大鼠Tscl甲基化表达与空白组无差异(P0.05)。(2)Tscl基因片段2甲基化：与对照组相比较,肥胖组大鼠Tscl基因片段2甲基化表达升高(P0.05)：电针干预治疗后,针刺组大鼠Tsc1基因片段2甲基化表达降低,低于肥胖组,但无明显差异(P0.05),针刺组大鼠Tscl基因片段2甲基化表达与空白组无差异(P0.05)。(3)Tsc1基因片段3甲基化：与对照组相比较,肥胖组大鼠Tsc1基因片段3甲基化表达显著性升高(P0.01)；电针干预治疗后,针刺组大鼠Tsc1基因片段3甲基化表达降低,低于肥胖组(P0.05),针刺组大鼠Tscl基因片段3甲基化表达与空白组无差异(P0.05)。3、下丘脑指标检测结果：(1)Dnmt2:与对照组相比较,肥胖组大鼠下丘脑Dnmt2平均光密度显著性升高(P0.01)；电针干预治疗后,针刺组大鼠下丘脑Dnmt2平均光密度明显降低,低于肥胖组(P0.01),针刺组大鼠下丘脑Dnmt2平均光密度与对照组无差异(P0.05)。(2)Dnmt3a:与对照组相比较,肥胖组大鼠下丘脑Dnmt3a平均光密度显著性升高(P0.01)；电针干预治疗后,针刺组大鼠下丘脑Dnmt3a平均光密度降低,低于肥胖组(P0.05),针刺组大鼠下丘脑Dnmt3a平均光密度与对照组无差异(P0.05)。 (3) Dnmt3b:与对照组相比较,肥胖组大鼠下丘脑Dnmt3b平均光密度显著性升高(P0.01)；电针干预治疗后,针刺组大鼠下丘脑Dnmt3b平均光密度明显降低,低于肥胖组(P0.01),针刺组大鼠下丘脑Dnmt3b平均光密度与对照组无差异(P0.05)。(4) mTORC1 mRNA和蛋白：与对照组相比较,肥胖组大鼠下丘脑mTORC1基因mRNA、蛋白显著性升高(P0.01)；电针干预治疗后,针刺组大鼠下丘脑mTORC1基因mRNA、蛋白明显降低,低于肥胖组(P=0.01,P0.05),针刺组大鼠下丘脑mTORC1基因mRNA、蛋白高于对照组(P0.05,P0.01)。(5) AgRP mRNA:与对照组相比较,肥胖组大鼠下丘脑AgRP基因mRNA显著性升高(P0.01)；电针干预治疗后,针刺组大鼠下丘脑AgRP基因mRNA明显降低,低于肥胖组(P0.01),针刺组大鼠下丘脑AgRP基因mRNA高于对照组,但无统计学差异(P0.05). (6) NPY mRNA:与对照组相比较,肥胖组大鼠下丘脑NPY基因mRNA显著性升高(P0.01)；电针干预治疗后,针刺组大鼠下丘脑NPY基因mRNA明显降低,低于肥胖组(P0.01),针刺组大鼠下丘脑NPY基因mRNA高于对照组(P0.01)。 (7) PoMC mRNA:与对照组相比较,肥胖组大鼠下丘脑PoMC基因mRNA显著性降低(P0.01)；针刺组大鼠下丘脑PoMC基因mRNA明显降低,高于肥胖组(P0.05),电针干预治疗后,针刺组大鼠下丘脑PoMC基因mRNA低于对照组(P0.05)。结论：1、针刺“中脘”、“天枢”、“三阴交”、“后三里”能有效减轻DIO大鼠体重,降低体脂含量及体脂率；2、DIO大鼠下丘脑DNA甲基化转移酶(Dnm2、Dnmt3a、Dnmt3b)活性增加,Tsc1基因启动子甲基化增加;针刺治疗单纯性肥胖,可能通过负调控DNA甲基化酶转移酶的活性,使Tsc1去甲基化,抑制mTORC1活性,从而调控食欲调节因子NPY、AgRP及PoMC等改善肥胖。
[Abstract]:Objective : To study diet - induced obesity . In this study , the effect and mechanism of acupuncture in the pathogenesis of obesity were observed .
The normal feed group was fed with common feed for 14 weeks , and the rats with no weight were removed as the control group . The acupuncture treatment was as follows : " Tianshu , Zhongwan , Sanjiao and Houshi " points were selected , and the rats were treated with electroacupuncture . The control group and the obese group were fixed without acupuncture . After the acupuncture treatment , the weight was weighed and the rats were fasted for 12 hours . After the acupuncture treatment , the body weight was weighed and the expression of Dnmts ( Dnm2 , Dnmt3a , Dnmt3b ) in the hypothalamic arcuate nucleus was detected by immunohistochemistry .
Western Blot was used to detect the protein expression of the hypothalamic arcuate nucleus mTORC1 gene in rats .
Results : 1 . Body weight : Before acupuncture intervention , the body weight of DIO rats in obesity group and acupuncture group was significantly higher than that in control group ( P0.01 ) , there was no statistical difference between obesity group and acupuncture group ( P0.05 ) .
Compared with the control group , the expression of Tscl methylation in the obese group was lower than that in the obese group ( P0.05 ) .
( 1 ) Dnmt2 : Compared with control group , the mean optical density of hypothalamus Dnmt2 in obese group was significantly higher than that in control group ( P0.01 ) .
( 2 ) Dnmt3a : Compared with the control group , the mean optical density of Dnmt3a in the hypothalamus of the obese group was significantly higher than that in the control group ( P0.01 ) .
( 3 ) Dnmt3b : Compared with the control group , the mean optical density of Dnmt3b in the hypothalamus of the obese group was significantly higher than that in the control group ( P0.01 ) .
( 4 ) mTORC1 mRNA and protein : Compared with control group , mTORC1 mRNA and protein increased significantly ( P0.01 ) .
Compared with the control group , the mRNA and protein of the mTORC1 gene mRNA and protein in the hypothalamus of the acupuncture group were significantly lower than those in the control group ( P0.05 , P0.01 ) . ( 5 ) AgRP mRNA : Compared with the control group , the mRNA of AgRP gene mRNA in the hypothalamus of the obese group was significantly increased ( P0.01 ) .
The mRNA of AgRP gene in hypothalamus of acupuncture group was significantly lower than that in obese group ( P0.01 ) . The mRNA of AgRP gene in hypothalamus of acupuncture group was higher than that of control group , but there was no statistical difference ( P0.05 ) .
The mRNA of NPY mRNA in hypothalamus of acupuncture group was significantly lower than that in obese group ( P0.01 ) . The mRNA of NPY mRNA in hypothalamus of acupuncture group was higher than that in control group ( P0.01 ) .
The mRNA of PoMC gene in hypothalamus of acupuncture group was significantly lower than that in obese group ( P0.05 ) . The mRNA of PoMC gene in hypothalamus of acupuncture group was lower than that of control group ( P0.05 ) . Conclusion : 1 . Acupuncture " Zhongwan " , " Tianshu " , " Three Yin Jiao " , " Three Ridges " can effectively reduce the weight of DIO rats , lower body fat content and body fat rate ;
2 . The activity of DNA methyltransferase ( Dnm2 , Dnmt3a , Dnmt3b ) in the hypothalamus of DIO rats increased , the methylation of Tsc1 gene promoter was increased , and the activity of DNA methylase transferase could be regulated by acupuncture .
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R245.97

【相似文献】

相关期刊论文 前10条

1 陈同度,张昌颖;素食大鼠的贫血现象[J];营养学报;1957年04期

2 陈伟强;赵善广;;自制注射用大鼠固定装置[J];上海实验动物科学;1992年04期

3 肖柳英,林培英,冯昭明,张丹;不同周龄的SD大鼠生理、生化及体重的正常值测定[J];中药新药与临床药理;1996年03期

4 李淑云;简易大鼠灌胃器的制作[J];锦州医学院学报;2001年04期

5 杨明智,陈积圣;一种大鼠抓取与固定的新工具介绍[J];上海实验动物科学;2001年03期

6 戴英,陆群;复方H_(505)对Wistar大鼠外周血的血液流变学指标的影响[J];中国血液流变学杂志;2001年01期

7 韦应波,孙喜庆,曹新生,姚永杰,冯岱雅,杨长斌;+Gz暴露时间对大鼠记忆功能和行为的影响[J];航天医学与医学工程;2003年01期

8 吕学军,郭俊生,李敏,周利梅,张永娟;晕船大鼠体内铁含量的变化[J];中国职业医学;2003年04期

9 汤仁仙,王迎伟,王慧,周峰;201A中药合剂对大鼠抗肾小球基底膜肾炎病变的影响[J];徐州医学院学报;2003年06期

10 孙同柱,付小兵,翁立新,梁雪梅,陈伟;介绍一种简易的大鼠保定方法[J];上海实验动物科学;2004年01期

相关会议论文 前10条

1 尹音;孙振宇;胡敏;李冬霞;;持续性高正加速度对大鼠颞颌关节损伤的作用[A];第八届全国颞下颌关节病学及（牙合）学大会论文汇编[C];2011年

2 祝~=骧;iJ梊霞;洃克琴;崔素英;文允摪;，

本文编号：1936276