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  本文選題：Neu-P11　切入點：褪黑素　出處：《南華大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景褪黑素(melatonin,Mel)是腦松果腺合成和分泌的一種吲哚類神經(jīng)內(nèi)分泌活性物質(zhì),具有調(diào)節(jié)晝夜節(jié)律、改善睡眠等功能。Neu-P11則是一種新型褪黑素非選擇性受體激動劑,我們前期的研究表明Neu-P11可調(diào)節(jié)高糖高脂高膽固醇誘導(dǎo)的肥胖大鼠糖脂代謝并改善其胰島素抵抗(insulin resistance,IR)狀態(tài),體外細胞試驗也證實Neu-P11能夠抑制甘油三酯聚積改善3T3-L1脂肪細胞IR。有研究報道,細胞內(nèi)活性氧(reactive oxygen species,ROS)水平過高造成的氧化應(yīng)激和線粒體功能障礙是導(dǎo)致胰島素抵抗的主要因素�；贜eu-P11、氧化應(yīng)激以及IR之間的聯(lián)系,本課題在前期實驗的基礎(chǔ)上提出假想:Neu-P11是否通過抑制氧化應(yīng)激而改善高糖高胰島素(high glucose and high insulin,HGI)誘導(dǎo)的3T3-L1脂肪細胞IR。方法(1)3T3-L1脂肪細胞的誘導(dǎo)及分化:經(jīng)典“雞尾酒”法(0.5mmol/L IBMX,1μmol/L DEX,10μg/ml INS)誘導(dǎo)前脂肪細胞分化為成熟3T3-L1脂肪細胞,油紅O染色鑒定脂肪細胞形態(tài)及3T3-L1前脂肪細胞的分化程度。(2)IR模型的制備:高糖(25 mmol/L)高胰島素(1μmol/L INS)孵育脂肪細胞24 h,葡萄糖氧化酶法檢測糖消耗量驗證胰島素抵抗細胞模型構(gòu)建是否成功。(3)熒光探針法、羅丹明123及葡萄糖氧化酶法分別檢測Neu-P11或Mel干預(yù)前后各組脂肪細胞中ROS含量、線粒體膜電位(mitochondrial transmembrane potential,MMP)變化及培養(yǎng)基內(nèi)葡萄糖含量。(4)Western Blot檢測細胞c-jun氨基末端激酶(Jun N-terminal kinase,JNK)、核轉(zhuǎn)錄蛋白p65(Nuclear factor kappa B p65,NF-κBp65)、蛋白激酶B(Protein kinase B,Akt)的表達變化及Neu-P11或Mel干預(yù)對上述蛋白表達的影響。結(jié)果3T3-L1前脂肪細胞經(jīng)“雞尾酒”法誘導(dǎo)10 d后通過油紅O染色可見90%以上細胞呈現(xiàn)明顯的“戒環(huán)樣”結(jié)構(gòu):細胞呈圓形,內(nèi)含大量脂滴,且呈環(huán)形排列,即表明已分化為典型的成熟脂肪細胞。實驗結(jié)果顯示:高糖高胰島素聯(lián)合作用3T3-L1脂肪細胞24 h能明顯降低細胞的葡萄糖消耗量,成功誘導(dǎo)其發(fā)生胰島素抵抗。另外,實驗結(jié)果還表明:與Control組相比,IR組細胞ROS含量明顯增高,MMP下降,細胞內(nèi)JNK/NF-κBp65蛋白表達增加,胰島素信號蛋白Akt表達下降。而經(jīng)Neu-P11處理可以明顯逆轉(zhuǎn)高糖高胰島素引起的生理效應(yīng),差異均具有統(tǒng)計學(xué)意義,且與Mel組作用相似。結(jié)論Neu-P11可以改善HGI誘導(dǎo)的3T3-L1脂肪細胞IR狀態(tài),其機制可能與Neu-P11清除ROS而改善線粒體功能、抑制JNK/NF-κBp65蛋白的活性有關(guān)。
[Abstract]:Background melatonin melatonin (melatonin) is an indole neuroendocrine active substance synthesized and secreted by the pineal gland of the brain. Neu-P11 is a novel melatonin nonselective receptor agonist with the functions of regulating circadian rhythm and improving sleep. Our previous studies have shown that Neu-P11 can regulate glucose and lipid metabolism and improve insulin resistance and insulin resistance in obese rats induced by high glucose, high fat and high cholesterol. In vitro cell tests also confirmed that Neu-P11 could inhibit the accumulation of triglycerides and improve the IR3 of 3T3-L1 adipocytes. Oxidative stress and mitochondrial dysfunction caused by excessive levels of reactive oxygen in cells are the main factors leading to insulin resistance, based on the relationship between Neu-P11, oxidative stress and IR. On the basis of previous experiments, this study proposed a hypothetical hypothesis that whether or not: Neu-P11 can improve the induction and differentiation of 3T3-L1 adipocytes induced by high glucose and high glucose and high insulin I by inhibiting oxidative stress. Methods the induction and differentiation of 3T3-L1 adipocytes: classic cocktail. "0.5 mmol / L IBM X1 渭 mol/L DEXX 10 渭 g / ml ins) induced adipocytes to differentiate into mature 3T3-L1 adipocytes. Identification of adipocyte morphology by oil red O staining and differentiation of 3T3-L1 preadipocytes. Preparation of IR model: hyperglycemic 25 mmol / L) hyperinsulinemia 1 渭 mol/L ins) incubated adipocytes for 24 h. Glucose oxidase assay was used to detect glucose consumption to verify insulin resistance. Whether the cell model was successfully constructed or not, the fluorescence probe method was used. Rhodamine 123 and glucose oxidase method were used to detect ROS content in adipocytes before and after Neu-P11 or Mel intervention. Changes of Mitochondrial transmembrane potentialMMPs and glucose content in the medium. Western Blot to detect the expression of c-jun amino-terminal kinases Jun N-terminal kinases, nuclear factor kappa Bp65 protein NF- 魏 Bp65, protein kinase B protein in kinase Bnktand Neu-P11 or Mel intervention on these proteins. Results the 3T3-L1 preadipocytes were induced by "cocktail" method for 10 days. Through oil red O staining, it was found that more than 90% cells showed "ring ring" structure: the cells were round. The results showed that 3T3-L1 adipocytes treated with high glucose and high insulin for 24 h could significantly reduce the glucose consumption of 3T3-L1 adipocytes. In addition, the results also showed that compared with Control group, the content of ROS increased significantly and the expression of JNK- / NF- 魏 Bp65 protein increased in IR group. The expression of insulin signal protein Akt was decreased, and the physiological effects induced by high glucose and high insulin could be reversed by Neu-P11 treatment, and the difference was statistically significant. Conclusion Neu-P11 can improve the IR state of 3T3-L1 adipocytes induced by HGI, and its mechanism may be related to Neu-P11 scavenging ROS and improving mitochondrial function and inhibiting the activity of Neu-P11 / NF- 魏 Bp65 protein.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.1

【參考文獻】

相關(guān)期刊論文 前7條

1 劉云濤;胡斌;簡磊;李建偉;黃亮;;血清網(wǎng)膜素-1與胰島素抵抗水平的相關(guān)性研究[J];中國全科醫(yī)學(xué);2012年33期

2 呂攀攀;瞿平;畢楊;陳潔;張蔓;劉友學(xué);;高脂飼料誘導(dǎo)的肥胖大鼠網(wǎng)膜素水平的實驗研究[J];生命科學(xué)研究;2012年02期

3 李健;余蘭;;糖尿病視網(wǎng)膜病變患者血漿網(wǎng)膜素的變化及其相關(guān)因素[J];中國老年學(xué)雜志;2012年08期

4 童國玉;胡云;沈山梅;陳煒;黃洪;朱大龍;;糖負荷對小鼠小腸網(wǎng)膜素基因表達的影響及網(wǎng)膜素對C2C12細胞胰島素增敏的機制[J];現(xiàn)代生物醫(yī)學(xué)進展;2012年08期

5 ;Berberine reverses free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ[J];World Journal of Gastroenterology;2008年06期

6 Ｓ．Ｆ．Ｐａｎｇ,Ｃ．Ｓ．Ｐａｎｇ,Ａ．Ｍ．Ｓ．Ｐｏｏｎ,Ｐ．Ｐ．Ｎ．Ｌｅｅ,Ｚ．Ｍ．Ｌｉｕ,Ｓ．Ｙ．Ｗ．Ｓｈｉｕ;Melatonin : a chemical photoperiodic signal with clinical significance in humans[J];Chinese Medical Journal;1998年03期

7 蔡潤策;魏麗;狄建忠;于浩泳;包玉倩;賈偉平;;肥胖和2型糖尿病患者脂肪組織網(wǎng)膜素的mRNA表達[J];中華醫(yī)學(xué)雜志;2009年06期

，

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