沉默PLIN1基因与杨梅素联合作用对3T3-L1细胞脂解的影响及机制探究

发布时间：2018-02-03 02:02

本文关键词： 杨梅素围脂滴蛋白 RNA干扰 3T3-L1脂肪细胞　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:采用杨梅素(Myricetin,Myric)与沉默围脂滴蛋白(Perilipin1,PLIN1)基因联合作用的方法,观察其对3T3-L1脂肪细胞脂解作用的影响,并进一步对其机制进行探究,为肥胖症的防治提供新的方向和思路。方法:1.常规培养及诱导分化3T3-L1前脂肪细胞。2.分别以0、1、5、10、50、100μmol/L的Myric浓度梯度以及48h和72h的时间梯度干预分化成熟的3T3-L1脂肪细胞,测定细胞中TG和甘油含量。3.根据筛选出的Myric最佳干预浓度联合sh-RNA高效转染载体对已诱导分化成熟的3T3-L1脂肪细胞进行联合干预实验,分为四组:联合干预组(Myric+sh-RNA)、转染组(sh-RNA)、杨梅素组(Myric)和空白组,测定细胞中TG和甘油含量,油红O染色,观察脂滴形态。4.联合干预后,采用Western blot检测PLIN1A、ATGL、HSL和p-HSL、ERK和p-ERK、MEK和p-MEK的表达量。采用双抗体夹心ABC-ELISA法检测细胞中cAMP、PKA和p-PKC的含量。结果:1.Myric干预浓度为100μmol/L,干预时间为72h时,细胞内TG含量最低,甘油含量最高,差异有统计学意义(P0.05)。2.联合干预后,Myric+sh-RNA组细胞内TG含量低于Myric组和sh-RNA组,甘油含量升高,差异有统计学意义(P0.05);Myric+sh-RNA组细胞内脂滴数量明显减少、形态明显变小。3.联合干预后,Myric+sh-RNA组PLIN1A蛋白表达量低于Myric组和sh-RNA组,ATGL和HSL蛋白表达量高于Myric组和sh-RNA组,差异有统计学意义(P0.05);Myric+sh-RNA组和Myric组p-HSL蛋白表达量高于sh-RNA组和空白组,差异有统计学意义(P0.05)。4.联合干预后,Myric+sh-RNA组和Myric组细胞内c AMP和PKA含量低于sh-RNA组和空白组,差异有统计学意义(P0.05);Myric+sh-RNA组和Myric组细胞内p-PKC含量,以及p-ERK/ERK、p-MEK/MEK的比值均大于sh-RNA组和空白组,差异有统计学意义(P0.05)。结论:1.Myric最佳干预浓度为100μmol/L,干预时间为72h。2.Myric与沉默PLIN1联合作用可以更大程度地提高脂解效率。3.Myric与沉默PLIN1联合作用可更有效降低PLIN1表达量,提高ATGL与HSL的表达量。4.Myric促进脂解作用可能是通过激活PKC-MEK/ERK信号通路,增加该通路中p-HSL表达量来实现的;Myric同时可能对cAMP/PKA信号通路中相关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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2

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