下调Fsp27基因表达联合杨梅素干预对3T3-L1细胞脂解的影响

目的:下调脂肪特异性蛋白27(Fsp27)基因表达联合杨梅素干预,观察对3T3-L1细胞中脂质代谢的影响,并探究脂滴发生、发展变化的调控机制。方法:常规培养3T3-L1前脂肪细胞,采用"鸡尾酒"法诱导其分化为成熟脂肪细胞。脂质体法转染sh-Fsp27干扰载体,以杨梅素浓度为100μmol/L的完全培养基干预成熟脂肪细胞72h。油红O染色,观察脂滴形态及大小的变化;酶法测定细胞内甘油及甘油三酯的含量,观察细胞脂质代谢的变化。Western blot检测Fsp27、激素敏感性甘油三酯脂肪酶(HSL)、甘油三酯脂肪酶(ATGL)以及丝裂原活化蛋白激酶(MAPK)信号通路蛋白的表达。结果:1. 3T3-L1细胞诱导分化后,形态由纤维样变成圆形,并伴随有细胞体积的增大。2.与对照组相比,杨梅素组和转染组细胞中甘油三酯含量下降,甘油含量升高(P 0. 05)。与其他三组相比,联合干预组细胞中甘油三酯含量减少,甘油含量增加(P 0. 05)。3.与对照组相比,其余三组细胞内Fsp27蛋白的表达量均降低,ATGL和PPARγ的表达量升高(P 0. 05)。另外,联合干预组和杨梅素组细胞内HSL的表达量和p-p38MAPK/p38MAPK的比值均大于sh-Fsp27组和对照组(P 0. 05)。结论:1. Fsp27基因沉默与杨梅素联合干预可以更大程度地促进脂肪分解代谢。2.杨梅素可通过激活MAPK信号通路,上调HSL和ATGL的蛋白表达来发挥其促脂解的作用; sh-Fsp27干扰载体通过调节PPARγ和Fsp27蛋白的表达,增加ATGL含量来加速脂肪分解。

The Effect and Mechanism of Down-Regulated Fsp27 Gene Combined with Myricetin on Lipolysis of 3T3-L1 Adipocytes

Objective: Using RNAi technology to reduce Fsp27 expression combined with Myric and observe the effect of lipid metabolism on 3T3-L1 cells, and explore the regulation mechanism of lipid droplets, development and changes.Methods: 3T3-L1 preadipocytes were routinely cultured and induced differentiation by the hormonal cocktail. The shRNA recombinant vectors were transfected into the differentiated 3T3-L1 adipocytes with lipofectamine. The medium was treated with Myric concentration of 100 μmol/L for 72 h. The oil red O staining photographed the morphology and sizes of lipid droplets. The content level of adipocytes cellular TG and glycerol were tested through enzymatic method to evaluate the lipolysis. Western blot measured the expression levels of Fsp27, the adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), p38MAPK, p-p38MAPK and PPARγ.Results: 1. After differentiation of 3T3-L1 cells, the morphology changed from fiber-like to round, accompanied by an increase in cell volume. 2. After combined intervention, compared to the control group, content of adipocytes cellular TG decreased with the increased glycerol in sh-Fsp27 group and Myric group (P＜0.05). Meanwhile, compared to the other three groups, the TG content in Myric+sh-Fsp27 group was lower, and the glycerol content increased (P＜0.05). 3. After combined intervention, compared to the control group, the expression levels of Fsp27 in the other three groups decreased significantly, and the expression levels of ATGL and PPARγ increased significantly (P＜0.05). In addition, the expression levels of HSL and p-p38MAPK/p38MAPK in Myric+sh-Fsp27 group and Myric group was higher than that in sh-Fsp27 group and control group (P＜0.05).Conclusions: 1. The combination of Fsp27 gene silencing and Myric could more obviously improve the efficiency of lipolysis than individual. 2. The regulating effect of Myric on lipolysis of 3T3-L1 adipocytes might be exerted through the MAPK pathway by up-regulating the level of ATGL and HSL. The sh-Fsp27 vectors could promote lipolysis by blocking Fsp27 gene and increasing the expression level of ATGL.