【摘要】 目的探讨3T3-L1脂肪细胞在营养剥夺状态下自噬的发生及自噬相关基因Beclin1的变化。方法培养3T3-L1脂肪细胞至不同诱导分化时期,对照组不做特别处理,营养剥夺组以Hanks液代替细胞完全培养液3 h,形成营养剥夺状态。采用透射电镜观察自噬体;转染携带微管相关蛋白1轻链3(LC3)的绿色荧光蛋白慢病毒(LC3-GFP-LENTIVIRAL)质粒系统,病毒扩增后感染3T3-L1靶细胞,荧光显微镜下观察LC3-GFP点状聚集;蛋白免疫印迹分析检测自噬抗体LC3以及Beclin1的变化;双荧光素酶报告基因检测Beclin1基因转录区(Promoter)及3′非编码区(3′UTR)的变化。结果营养剥夺组自噬体数量较对照组增多;营养剥夺组较对照组LC3-GFP点状聚集增多;营养剥夺组LC3-Ⅱ、Beclin1蛋白表达较对照组升高;与对照组相比,营养剥夺组Beclin1-Promoter及Beclin1-3′UTR相对荧光单位呈倍数增高,差别有统计学意义(P<0.01)。结论营养剥夺可以诱导3T3-L1脂肪细胞发生自噬;营养剥夺诱导的自噬与Beclin1相关,可同时影响Beclin1基因的转录与mRNA稳定性。更多还原

【Abstract】 Objective To study the nutrient deprivation induced autophagy in 3T3-L1 adipocytes and the alteration of Beclin1.Methods The 3T3-L1 adipocytes were cultured to different stages,and the control group without any special treatments.The nutrient deprivation stage was formed by use of Hank′s balanced salt solution for 3 hours in nutrient deprivation group.Autophagosome was observed by transmission electron microscope.The LC3-GFP-LENTIVIRAL plasmid systems was transfected,and the 3T3-L1 target cells were infected after amplification of virus,then the LC3-GFP point-like aggregation was observed by fluorescence microscope;the change of LC3 and Beclin1 were detected by western blot;the changes of Beclin1-Promoter and 3′UTR were detected by dual-luciferase reporter gene.Results The autophagosomes and LC3-GFP Point-like aggregation in nutrient deprivation group were more than those in control.The expression of LC3-Ⅱ and Beclin1 were increased in nutrient deprivation group compared with control group.The Beclin1-Promoter and Beclin1-3′UTR increased exponentially in nutrient deprivation group compared with control group(P<0.01).Conclusion Nutrient deprivation could induce autophagy in 3T3-L1 adipocytes.Autophagy induced by nutritional deprivation associated with Beclin1,which can affect gene transcription of Beclin1 and the stability of mRNA.更多还原