小檗碱对脂肪胰岛素抵抗细胞PPARγ及脂肪分泌因子表达影响.docVIP

小檗碱对脂肪胰岛素抵抗细胞PPARγ及脂肪分泌因子表达影响 　　[摘要] 脂肪分泌细胞因子与脂肪组织胰岛素抵抗（IR）密切相关，在糖尿病的发生发展研究中越来越受到重视。该实验主要研究小檗碱对脂肪胰岛素抵抗（IR）细胞PPARγ和脂肪分泌因子mRNA表达的影响，探讨小檗碱增强胰岛素敏感性的分子机制及微滴数字PCR（ddPCR）方法的应用优势。该实验采用ddPCR绝对定量分析简单直观确定合适的内参基因，ddPCR和荧光定量法（qPCR）方法比较研究不同剂量（10，20，50，100 μmol?L-1）小檗碱干预IR 3T3-L1脂肪细胞中PPARγ、脂联素、抵抗素和瘦素mRNA表达；加入GW9662研究PPARγ和脂联素mRNA表达的内在关联。ddPCR结果发现β-actin 在脂肪细胞中表达稳定，适合作为内参基因对定量PCR数据标准化。ddPCR和qPCR方法均显示与IR模型组相比，10，20，50，100 μmol?L-1小檗碱呈现剂量依赖降低脂联素mRNA表达（P-1小檗碱呈现剂量依赖降低其表达（P-1降低其表达（P0.05）。PPARγ特异拮抗剂GW9662干预实验表明脂联素基因表达受PPARγ直接调控（P0.05）。ddPCR探针法检测到各剂量小檗碱均剂量依赖显著降低抵抗素和瘦素mRNA表达（P0.01）。结果揭示小檗碱调节脂联素起到胰岛素增敏作用并非通过PPARγ依赖途径在转录调控水平上调脂联素表达，推测可能是在翻译后调控水平上调高聚体脂联素比例。小檗碱下调抵抗素和瘦素表达减轻脂解改善IR。ddPCR比qPCR具有更好线性范围和灵敏度，对低丰度表达基因检测具有明显的技术优势。 　　[关键词]小檗碱； 胰岛素抵抗； PPARγ； 脂肪细胞因子； 微滴数字PCR（ddPCR） 　　[Abstract] Adipocytokines are closely associated with insulin resistance （IR） in adipose tissues， and they are more and more seriously taken in the study of the development of diabetes. This experiment was mainly to study the effect of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin resistant adipocytes， and investigate the molecular mechanism of berberine in enhancing insulin sensitization and application advantages of droplet digital PCR （ddPCR）. ddPCR absolute quantification analysis was taken in this experiment to simply and intuitively determine the appropriate reference genes. ddPCR and quantitative Real-time PCR （qPCR） were used to compare the effect of different doses of berberine （10， 20， 50， 100 μmol?L-1） on mRNA expression levels of PPARγ， adiponectin， resistin and leptin in IR 3T3-L1adipocytes. Antagonist GW9662 was added to study the inherent correlation between PPARγ and adiponectin mRNA expression levels. ddPCR results showed that the expression level of β-actin in adipocytes was stable， and suitable as reference gene for normalization of quantitative PCR data. Both of ddPCR and qPCR results showed that， as compared with IR models， the mRNA expression levels of adiponectin were de