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本文旨在通过观察前脂肪细胞分化过程中促酰化蛋白(acylation stimulating protein,ASP)对脂滴相关蛋白adipophilin和perilipin表达的影响,探讨adipophilin和perilipin在介导ASP促成脂过程中的作用。体外培养小鼠来源的3T3-L1前脂肪细胞,经典激素鸡尾酒法诱导分化,根据实验室前期工作验证的adipophilin和perilipin基因和蛋白水平的时序性变化规律选取几个代表性的时间点(0d,3d,6d,9d),设立ASP(1μmol/L)刺激组、胰岛素(100nmol/L)刺激组及空白对照组,[3H]油酸掺入法测定脂肪细胞甘油三酯合成率,[3H]-2-脱氧葡萄糖([3H]-2-DG)掺入法测定脂肪细胞葡萄糖转运率,RT-PCR和Western blot方法分别检测adipophilin和perilipin mRNA和蛋白表达。结果显示:(1)脂肪细胞分化第3天和第6天,ASP组甘油三酯合成率显著高于空白对照组(P<0.05,P<0.01),胰岛素组各检测时间点甘油三酯合成率与正常对照组之间无显著性差异;(2)脂肪细胞分化第6天和第9天,ASP组和胰岛素组葡萄糖转运率均较同期空白对照组显著增加(P<0.05,P<0.01),胰岛素组增加效应较ASP组更为显著(P<0.05,P<0.001);(3)与空白对照组相比,ASP组adipophilin基因和蛋白表达水平从0天起显著升高(P<0.05,P<0.001),第4天以后无显著统计学差异,ASP组perilipin基因和蛋白表达水平从第3天起显著升高(P<0.05,P<0.001),直至完全分化成熟(第9天)仍显著高于同期对照组(P<0.05);(4)胰岛素组adipophilin和perilipin基因和蛋白表达水平均与同期对照组相比无显著统计学差异。以上结果提示,ASP组perilipin及adipophilin的表达与脂肪细胞分化过程中ASP促进甘油三酯合成之间具有显著相关性,ASP可能通过调节adipophilin、perilipin基因和蛋白的表达影响脂肪细胞内脂质聚积和脂滴形成。
This study aimed to investigate the effects of acylation stimulating protein (ASP) on the expression of adipophilin and perilipin in adipocytes and to explore the role of adipophilin and perilipin in mediating ASP-induced lipids. The mouse-derived 3T3-L1 preadipocytes were cultured in vitro and were induced to differentiate by classical hormone cocktail method. Several representative time points were selected according to the regularity of adipophilin and perilipin gene and protein levels (0d, 3 h, 6 d, 9 d). The triglyceride synthesis rates of adipocytes were determined by [3H] oleic acid incorporation assay with [1 H] ASP and 100 nmol / The glucose transport rate of adipocytes was measured by incorporation of [3H] -2-DG. The mRNA and protein expression of adipophilin and perilipin were detected by RT-PCR and Western blot respectively. The results showed that: (1) The rate of triglyceride synthesis in ASP group was significantly higher than that in the blank control group (P <0.05, P <0.01) on the 3rd and 6th day of adipocyte differentiation. The triglyceride synthesis (2) On day 6 and day 9 of adipocyte differentiation, the glucose transport rates of ASP group and insulin group were significantly higher than those of the blank control group (P <0.05, P <0.01) ), Insulin group increased more significantly than ASP group (P <0.05, P <0.001); (3) compared with the blank control group, ASP group adipophilin gene and protein expression levels increased significantly from 0 days (P < (P <0.05, P <0.001, P <0.001). There was no significant difference after 4 days. The gene and protein expression of perilipin in ASP group were significantly increased from the 3rd day (P <0.05, P <0.001) Day) was still significantly higher than the control group at the same period (P <0.05). (4) The levels of adipophilin and perilipin mRNA and protein in the insulin group were not significantly different from those in the control group at the same period. These results suggest that perilipin and adipophilin expression in ASP group and ASP in adipocyte differentiation promote the synthesis of triglycerides was significantly correlated, ASP may regulate adipophilin, perilipin gene and protein expression affect adipocyte lipid accumulation and Lipid formation.