本文關(guān)鍵詞： 楊梅素 圍脂滴蛋白 RNA干擾 3T3-L1脂肪細(xì)胞　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:采用楊梅素(Myricetin,Myric)與沉默圍脂滴蛋白(Perilipin1,PLIN1)基因聯(lián)合作用的方法,觀察其對3T3-L1脂肪細(xì)胞脂解作用的影響,并進(jìn)一步對其機制進(jìn)行探究,為肥胖癥的防治提供新的方向和思路。方法:1.常規(guī)培養(yǎng)及誘導(dǎo)分化3T3-L1前脂肪細(xì)胞。2.分別以0、1、5、10、50、100μmol/L的Myric濃度梯度以及48h和72h的時間梯度干預(yù)分化成熟的3T3-L1脂肪細(xì)胞,測定細(xì)胞中TG和甘油含量。3.根據(jù)篩選出的Myric最佳干預(yù)濃度聯(lián)合sh-RNA高效轉(zhuǎn)染載體對已誘導(dǎo)分化成熟的3T3-L1脂肪細(xì)胞進(jìn)行聯(lián)合干預(yù)實驗,分為四組:聯(lián)合干預(yù)組(Myric+sh-RNA)、轉(zhuǎn)染組(sh-RNA)、楊梅素組(Myric)和空白組,測定細(xì)胞中TG和甘油含量,油紅O染色,觀察脂滴形態(tài)。4.聯(lián)合干預(yù)后,采用Western blot檢測PLIN1A、ATGL、HSL和p-HSL、ERK和p-ERK、MEK和p-MEK的表達(dá)量。采用雙抗體夾心ABC-ELISA法檢測細(xì)胞中cAMP、PKA和p-PKC的含量。結(jié)果:1.Myric干預(yù)濃度為100μmol/L,干預(yù)時間為72h時,細(xì)胞內(nèi)TG含量最低,甘油含量最高,差異有統(tǒng)計學(xué)意義(P0.05)。2.聯(lián)合干預(yù)后,Myric+sh-RNA組細(xì)胞內(nèi)TG含量低于Myric組和sh-RNA組,甘油含量升高,差異有統(tǒng)計學(xué)意義(P0.05);Myric+sh-RNA組細(xì)胞內(nèi)脂滴數(shù)量明顯減少、形態(tài)明顯變小。3.聯(lián)合干預(yù)后,Myric+sh-RNA組PLIN1A蛋白表達(dá)量低于Myric組和sh-RNA組,ATGL和HSL蛋白表達(dá)量高于Myric組和sh-RNA組,差異有統(tǒng)計學(xué)意義(P0.05);Myric+sh-RNA組和Myric組p-HSL蛋白表達(dá)量高于sh-RNA組和空白組,差異有統(tǒng)計學(xué)意義(P0.05)。4.聯(lián)合干預(yù)后,Myric+sh-RNA組和Myric組細(xì)胞內(nèi)c AMP和PKA含量低于sh-RNA組和空白組,差異有統(tǒng)計學(xué)意義(P0.05);Myric+sh-RNA組和Myric組細(xì)胞內(nèi)p-PKC含量,以及p-ERK/ERK、p-MEK/MEK的比值均大于sh-RNA組和空白組,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1.Myric最佳干預(yù)濃度為100μmol/L,干預(yù)時間為72h。2.Myric與沉默PLIN1聯(lián)合作用可以更大程度地提高脂解效率。3.Myric與沉默PLIN1聯(lián)合作用可更有效降低PLIN1表達(dá)量,提高ATGL與HSL的表達(dá)量。4.Myric促進(jìn)脂解作用可能是通過激活PKC-MEK/ERK信號通路,增加該通路中p-HSL表達(dá)量來實現(xiàn)的;Myric同時可能對cAMP/PKA信號通路中相關(guān)因子的活性有一定的抑制作用。
[Abstract]:Objective: to study the effect of Myricetin (Myricin) and perilipin1 (PLIN1) gene on myricetin. To observe its effect on the lipid hydrolysis of 3T3-L1 adipocytes and explore its mechanism. Methods: 1. Conventional culture and differentiation of 3T3-L1 preadipocytes. The Myric concentration gradient of 100 渭 mol/L and the time gradient of 48 h and 72 h intervened the mature 3T3-L1 adipocytes. TG and glycerol contents in the cells were measured .3.According to the best intervention concentration of Myric and high efficient transfection vector of sh-RNA, the 3T3-L1 adipocytes were induced to differentiate and mature. Pretest. They were divided into four groups: the combined intervention group (Myric sh-RNAN), the transfection group (sh-RNAN), the myricin group (Myrica) and the blank group. The contents of TG and glycerol in the cells were measured. Oil red O staining was used to observe the morphology of lipid droplets. After combined intervention, Western blot was used to detect HSL, p-HSLERK and p-ERK. The expression of MEK and p-MEK was detected by double antibody sandwich ABC-ELISA method. Results 1. When the concentration of Myric was 100 渭 mol / L and the time of intervention was 72 h, the content of TG was the lowest and the content of glycerol was the highest. After combined intervention, the intracellular TG content in Myric sh-RNA group was lower than that in Myric group and sh-RNA group, and glycerol content was increased. The difference was statistically significant (P 0.05). In Myric sh-RNA group, the number of lipid droplets in the cells was significantly decreased, and the morphology was significantly reduced. 3. After combined intervention, the number of lipid droplets was significantly decreased. The expression of PLIN1A protein in Myric sh-RNA group was lower than that in Myric group and sh-RNA group. The expression of ATGL and HSL protein was higher than that of Myric group and sh-RNA group, and the difference was statistically significant (P 0.05). The expression of p-HSL protein in Myric sh-RNA group and Myric group was higher than that in sh-RNA group and blank group. The contents of c AMP and PKA in Myric sh-RNA group and Myric group were lower than those in sh-RNA group and blank group (P 0.05). The content of p-PKC and the ratio of p-ERK / ERK / MEK in Myric sh-RNA group and Myric group were higher than those in sh-RNA group and blank group. Conclusion: 1. The best intervention concentration of Myric is 100 渭 mol/L. Intervention time is 72h.2.Myric combined with silencing PLIN1 can improve the efficiency of lipid hydrolysis to a greater extent. 3.Myric combined with silent PLIN1 can reduce PLI more effectively. N1 expression. Increasing the expression of ATGL and HSL. 4. Myric promotes liposysis by activating the PKC-MEK/ERK signaling pathway and increasing the expression of p-HSL in the pathway. Myric may also inhibit the activity of related factors in cAMP/PKA signaling pathway.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R589.2

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