本文選題：電針 + 單純性肥胖　； 參考：《成都中醫(yī)藥大學》2016年碩士論文

【摘要】：目的：本研究以食源性肥胖(diet-induced obesity. DIO)大鼠模型為研究載體,從單純性肥胖(simple obesity)發(fā)病的關鍵環(huán)節(jié)-下丘腦Tsc1-mTOR信號通路入手,在前期本研究組證實DIO大鼠下丘腦結節(jié)性硬化癥因子1 (Tuberous sclerosis complex 1,Tscl)基因甲基化增加的基礎上,觀察下丘腦DNA甲基化轉移酶(DNAMthyl-Transferases, Dnmts)對Tsc1基因的甲基化修飾作用,調控下游哺乳動物雷帕霉素靶蛋白復合物1 (mammalian target of rapamycin complex 1, mTORC 1)及關鍵因子在單純性肥胖發(fā)病中的機制,從表觀遺傳學的角度觀察針刺治療肥胖的效應及機制。方法：選用剛斷乳的健康清潔級,4周齡(體重70-90g)雄性SD(Sprague-Dawley)大鼠,剔除體重不符合要求的大鼠,然后隨機分為造模組和普通飼料組。造模組運用改良后的高脂飼料喂養(yǎng)誘導DIO大鼠模型,造模14周后,將造模成功的DIO大鼠隨機分為肥胖組(即模型組)和針刺組；普通飼料組用普通飼料喂養(yǎng)14周,剔除體重不符合要求的大鼠后作為對照組。針刺治療：選取“天樞、中脘、三陰交和后三里”穴,用自制大鼠固定器固定大鼠后進行電針治療,選疏密波,2/15Hz,30min/天,5天／療程,休息2天,共4個療程,對照組和肥胖組同法固定但不針刺。針刺治療結束后稱量體重并予以禁食12h,于次日取動物標本,用免疫組化標記法檢測大鼠下丘腦弓狀核Dnmts (Dnm2、Dnmt3a、Dnmt3b)的表達情況；用Western Blot蛋白質印跡法檢測大鼠下丘腦弓狀核mTORC1基因的蛋白表達情況；運用RT-PCR對大鼠下丘腦弓狀核mTORC1及食欲肽(AgRP、NPY和PoMC) mRNA的含量及Tscl基因甲基化進行檢測,分別比較各組結果差異。結果：1、體重結果：針刺干預前,肥胖組和針刺組DIO大鼠體重明顯高于對照組(P0.01),肥胖組和針刺組組間體重無統(tǒng)計學差異(P0.05)；針刺干預治療后,肥胖組和針刺組體重、體脂及體脂率均明顯降低(P0.01)。2、下丘腦Tscl甲基化結果：(1)Tsc1片段1甲基化：與對照組相比較,肥胖組大鼠Tscl甲基化表達升高(P0.05)：電針干預治療后,針刺組大鼠Tsc1甲基化表達降低,低于肥胖組(P0.05),針刺組大鼠Tscl甲基化表達與空白組無差異(P0.05)。(2)Tscl基因片段2甲基化：與對照組相比較,肥胖組大鼠Tscl基因片段2甲基化表達升高(P0.05)：電針干預治療后,針刺組大鼠Tsc1基因片段2甲基化表達降低,低于肥胖組,但無明顯差異(P0.05),針刺組大鼠Tscl基因片段2甲基化表達與空白組無差異(P0.05)。(3)Tsc1基因片段3甲基化：與對照組相比較,肥胖組大鼠Tsc1基因片段3甲基化表達顯著性升高(P0.01)；電針干預治療后,針刺組大鼠Tsc1基因片段3甲基化表達降低,低于肥胖組(P0.05),針刺組大鼠Tscl基因片段3甲基化表達與空白組無差異(P0.05)。3、下丘腦指標檢測結果：(1)Dnmt2:與對照組相比較,肥胖組大鼠下丘腦Dnmt2平均光密度顯著性升高(P0.01)；電針干預治療后,針刺組大鼠下丘腦Dnmt2平均光密度明顯降低,低于肥胖組(P0.01),針刺組大鼠下丘腦Dnmt2平均光密度與對照組無差異(P0.05)。(2)Dnmt3a:與對照組相比較,肥胖組大鼠下丘腦Dnmt3a平均光密度顯著性升高(P0.01)；電針干預治療后,針刺組大鼠下丘腦Dnmt3a平均光密度降低,低于肥胖組(P0.05),針刺組大鼠下丘腦Dnmt3a平均光密度與對照組無差異(P0.05)。 (3) Dnmt3b:與對照組相比較,肥胖組大鼠下丘腦Dnmt3b平均光密度顯著性升高(P0.01)；電針干預治療后,針刺組大鼠下丘腦Dnmt3b平均光密度明顯降低,低于肥胖組(P0.01),針刺組大鼠下丘腦Dnmt3b平均光密度與對照組無差異(P0.05)。(4) mTORC1 mRNA和蛋白：與對照組相比較,肥胖組大鼠下丘腦mTORC1基因mRNA、蛋白顯著性升高(P0.01)；電針干預治療后,針刺組大鼠下丘腦mTORC1基因mRNA、蛋白明顯降低,低于肥胖組(P=0.01,P0.05),針刺組大鼠下丘腦mTORC1基因mRNA、蛋白高于對照組(P0.05,P0.01)。(5) AgRP mRNA:與對照組相比較,肥胖組大鼠下丘腦AgRP基因mRNA顯著性升高(P0.01)；電針干預治療后,針刺組大鼠下丘腦AgRP基因mRNA明顯降低,低于肥胖組(P0.01),針刺組大鼠下丘腦AgRP基因mRNA高于對照組,但無統(tǒng)計學差異(P0.05). (6) NPY mRNA:與對照組相比較,肥胖組大鼠下丘腦NPY基因mRNA顯著性升高(P0.01)；電針干預治療后,針刺組大鼠下丘腦NPY基因mRNA明顯降低,低于肥胖組(P0.01),針刺組大鼠下丘腦NPY基因mRNA高于對照組(P0.01)。 (7) PoMC mRNA:與對照組相比較,肥胖組大鼠下丘腦PoMC基因mRNA顯著性降低(P0.01)；針刺組大鼠下丘腦PoMC基因mRNA明顯降低,高于肥胖組(P0.05),電針干預治療后,針刺組大鼠下丘腦PoMC基因mRNA低于對照組(P0.05)。結論：1、針刺“中脘”、“天樞”、“三陰交”、“后三里”能有效減輕DIO大鼠體重,降低體脂含量及體脂率；2、DIO大鼠下丘腦DNA甲基化轉移酶(Dnm2、Dnmt3a、Dnmt3b)活性增加,Tsc1基因啟動子甲基化增加;針刺治療單純性肥胖,可能通過負調控DNA甲基化酶轉移酶的活性,使Tsc1去甲基化,抑制mTORC1活性,從而調控食欲調節(jié)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 .
【學位授予單位】：成都中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R245.97

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