摘要目的 探讨11β-羟基类固醇脱氢酶(11β-HSD1)与S100A16基因共同调节3T3-L1前脂肪细胞分化的作用及其机制.方法 分别构建慢病毒载体PUM1-11β HSD1和PLJM1-S100A16-GFP,共转染3T3-L1前脂肪细胞,用2.5 μg/ml嘌呤霉素筛选2周后建立11β-HSD1与S100A16共表达的稳定转染细胞株.以Western印迹法验证慢病毒载体转染效果,采用实时定量PCR检测脂肪细胞分化相关标志基因表达的变化,采用油红染色观察各组脂肪细胞分化后脂滴堆积水平,同时测定甘油三酯含量;以荧光素酶报告基因检测11β-HSD1与S100A16对PPARγ启动子活性影响.结果 11β-HSD1与S100A16蛋白在慢病毒感染的3T3-L1共转细胞株中的表达较空载对照细胞明显升高.在11β-HSD1与S100A16共转染3T3-L1细胞株诱导分化8d后,可见脂滴形成及甘油三酯含量较11β-HSD1与S100A16单独过表达的3T3-L1细胞株、正常3T3-L1及空载对照组明显升高(P＜0.05),脂肪细胞分化标志基因PPARγ、CCAAT增强子结合蛋白α(C/EBPα)、脂蛋白脂酶、脂肪细胞脂肪酸结合蛋白、脂肪酸合成酶表达明显上调.报告基因结果显示11β-HSD1与S100A16共表达明显增强PPARγ启动子活性.结论 11β-HSD1与S100A16可能通过协同调控PPARγ表达共同促进3T3-L1前脂肪细胞分化,在肥胖的发生发展中起重要作用.

abstractsObjective To investigate the synergistic effect of 11 β-hydroxysteriod dehydrogenase (11 β-HSD1) and S100A16 on the differentiation of3T3-L1 preadipocytes and its mechanism.Methods Lentiviral vectors PLJM1-11β-HSD1 and PLJM1-S100A16-GFP were respectively constructed and co-transfected into 3T3-L1 preadipocytes.The cell strains expressing 11 β-HSD1/S100A16 were screened with 2.5 μg/ml puromycin for two weeks.Western blot was employed to verify the lentiviral carrier transfection effects.The expressions of marker genes related to the adipocyte differentiation were detected by mean of realtime PCR.Oil red O staining was used to observe the lipid droplet accumulation and the content of triglyceride was measured after differentiation of preadipocytes.The effect of 11β-HSD1 and S100A16 on PPARγ promoter activity was detected by luciferase reporter gene.Results Compared with the empty vector group,the expressions of 11β-HSD1 and S100A16 protein in the lentivirus cotransfected 3T3-L1 cell strain were significantly higher.After 3T3-L1 cell strain co-expressing 1 1β-HSD1 and S100A16 was induced to differentiate for 8 days,the lipid droplets accumulation and triglyceride content were siginificantly increased,along with increased expressions of adipocyte differentiation marker genes such as PPARγ,CCAAT/enhancer binding protein α,lipoprotein lipase,fatty acid synthase,and adipocyte fatty acid-binding protein,in comparison with 11 β-HSD1 or S100A16 overexpression.The result of reporter gene indicated that 11 β-HSD1/ S100A16 enhanced PPARγ promoter activity.Conclusions 11β-HSD1 and S100A16 may jointly promote the differentiation of 3T3-L1 preadipocytes through a synergistic effect on PPARγexpression and play a critical role in the development of obesity.