沉默PLIN1基因与异丙肾上腺素对3T3-L1脂肪细胞脂解的机制探究

目的 研究沉默PLIN1基因与异丙肾上腺素(ISO)联合作用对3T3-L1脂肪细胞脂解的影响及机制探讨。方法 sh-PLIN1干扰载体成功转染后,以浓度为10μM的ISO处理3T3-L1脂肪细胞。采用油红O进行脂滴染色,观察脂滴形态;采用酶学方法测定甘油三酯(TG)和甘油含量,了解细胞脂解情况;采用Western-Blot法检测脂肪细胞中围脂滴蛋白A(PLIN1A)、脂肪甘油三酯脂肪酶(ATGL)、激素敏感性脂肪酶(HSL)和其磷酸化蛋白(p-HSL)的表达水平;双抗体夹心ABC-ELISA法检测细胞中环磷酸腺苷(cAMP)和蛋白激酶A(PKA)的浓度。结果 与空白组比较,ISO+sh-PLIN1组和ISO组脂滴变小,TG含量降低,甘油含量升高,有统计学意义(P<0.01)。同时,ISO+sh-PLIN1组和 sh-PLIN1组中ATGL 表达量均升高,有统计学意义(P<0.05)。ISO+sh-PLIN1转染组和ISO组中HSL、p-HSL、cAMP及 PKA 的表达量均上调(P<0.05)。结论 ISO促进脂解作用可能是cAMP/PKA通路介导,增加HSL和p-HSL的表达量来实现,而cAMP/PKA信号通路不能介导沉默PLIN1的促脂解作用。

Combined effect of PLIN1 gene silencing and isoproterenol on lipolysis of 3T3-L1 adipocytes

Objective To investigate the effect of combined intervention with isoproterenol and sh-PLIN1 on lipolysis of 3T3-L1 adipocytes,and to obtain insights into its molecular mechanism. Methods After sh-PLIN1 was transfected into 3T3-L1 adipocytes,10μmol/L isoproterenol (ISO) were used to treat adipocytes for 6 hours.The content level of adipocytes cellular TG and glycerol were tested through enzymatic method to evaluate the lipolysis.Western-Blot measured contents of the adipose triglyceride lipase (ATGL),hormone sensitive lipase (HSL) and phosphorylase-HSL (p-HSL).The contents of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) were detected by ABC-ELISA method. Results Compared to the negative control group,lipid droplets size and TG content decreased significantly with the increased glycerol（P<0.01）in ISO+sh-PLIN1 group and ISO group.The protein expression level of ATGL significantly increased in ISO+sh-PLIN1 group and sh-PLIN1 group（P<0.05).Moreover,the level of HSL,p-HSL,cAMP and PKA increased in ISO+sh-PLIN1 group and ISO group (P<0.05). Conclusion The regulating effect of ISO on lipolysis of 3T3-L1 adipocytes might be exerted through the cAMP/PKA pathway,which had no effect on PLIN1 gene silencing on it.