【作者】 王国强；

【导师】 尚文斌；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2011， 硕士

【摘要】 “脂肪细胞游离脂肪酸外溢学说”是2型糖尿病的重要发病机制之一,是开发糖尿病治疗药物的重要理论依据。本课题在前期的研究基础上,借助体外脂肪细胞培养和体内肥胖模型小鼠实验,以游离脂肪酸(free fatty acid, FFA)外溢为切入点,研究抗糖尿病药物人参的有效成分人参皂苷Rbl的作用机制。本课题研究内容包括人参皂苷Rbl对脂肪细胞基础状态以及胰岛素抵抗状态下FFA外溢水平的影响,观察人参皂苷Rbl对脂肪分解的影响以及机制,阐明人参治疗糖尿病的作用机制。有助于加深对人参抗糖尿病作用机制的系统认识和理解,为进一步研究抗糖尿病中药提供新的研究方向。目的：观察人参皂苷Rb1对体外培养的3T3-L1脂肪细胞和高脂喂养肥胖小鼠的影响,探明人参Rb1对脂肪细胞游离脂肪酸溢出的影响,探明其作用机制。方法：(1)观察人参皂苷Rbl对基础状态及胰岛素抵抗状态下3T3-L1脂肪细胞脂肪分解的影响；(2)用免疫印迹法观察人参皂苷Rb1对胰岛素抵抗状态下3T3-L1脂肪细胞中Perilipin表达影响；(3)激光共聚焦显微镜下观察人参皂苷Rb1对基础状态下3T3-L1脂肪细胞中Perilipin蛋白转位的影响；(4)观察人参皂苷Rb1对高脂饮食诱导的C57BL／6肥胖小鼠体重、附睾脂肪、肝脏重量及血清NEFA, TC, TG, HDL-C, LDL-C, TNF-a, IL-6水平的影响；人参皂苷Rb1对肥胖小鼠胰岛素敏感性的影响；人参皂苷Rbl对肥胖小鼠肝脏的病理形态学影响；人参皂苷Rbl对肥胖小鼠附睾脂肪中HSL的表达及HSL(Ser563)磷酸化表达水平的影响。结果：(1)人参皂苷Rbl没有显著地抑制基础状态下3T3-L1脂肪细胞脂解,亦没有明显对抗TNF-a诱导3T3-L1脂肪细胞脂解的增加；(2) TNF-a诱导分化成熟的3T3-L1脂肪细胞24小时引起Perilipin表达显著下降,同时用人参皂苷Rbl干预24小时后对于Perilipin的表达无显著影响,但是对于TNF-a所导致Perilipin表达下调有一定对抗作用；(3)人参皂苷Rbl促进基础状态下3T3-L1脂肪细胞内Perilipin由胞浆向脂滴表面的转位；(4)人参皂苷Rb1显著降低肥胖小鼠的附睾脂肪含量与体重比值；人参皂苷Rbl没有显著降低血清NEFA,对TC, TG, HDL-C, LDL-C无显著影响；人参皂苷Rbl可以明显提高改善肥胖小鼠胰岛素敏感性,改善胰岛素抵抗；模型组小鼠血清TNF-α、IL-6含量高于正常组,人参皂苷Rbl治疗后,TNF-α、IL-6水平均明显下降,与模型组比较有显著性差异：人参皂苷Rbl可以显著改善肥胖小鼠肝脏脂肪变性,减少肝细胞内脂肪的异位沉积；人参皂苷Rbl抑制肥胖小鼠附睾脂肪中HSL(Ser563)磷酸化表达,对HSL表达无明显影响。结论：(1)人参皂苷Rbl可能在一定水平上抑制3T3-L1脂肪细胞基础水平的脂肪分解；(2)人参皂苷Rbl可能能够对抗TNF-α所导致3T3-L1脂肪细胞的脂解增加；(3)人参皂苷Rb1可能通过促进脂肪细胞内Perilipin由胞浆向脂滴表面的转位而抑制基础状态下脂解；可能通过对抗TNF-α所导致Perilipin表达下调而抑制胰岛素抵抗状态的脂解；(4)人参皂苷Rb1可能在一定程度上降低游离脂肪酸水平,降低肥胖小鼠体内脂肪含量,改善脂质代谢,改善肥胖小鼠胰岛素抵抗,抑制TNF-α、IL-6等炎症因子水平；人参皂苷Rbl减少肝细胞内脂肪的异位沉积；人参皂苷Rb1可能通过抑制肥胖小鼠附睾脂肪中HSL(Ser563)磷酸化而抑制脂解。更多还原

【Abstract】 "Free fatty acid overflow hypothesis in adipocyte"—an important pathogenesis of type 2 diabetes, is used as an important target of drug treatment of diabetes mellitus development. Basing on previous research, taking free fatty acid overflow as the contact point, adipocyte cell culture in Vivo and obese model mice experiments in vitro were used to study the mechanism of ginsenoside Rb1, which is the active ingredient of anti-diabetic drugs ginseng. Using laser scanning confocal microscope, Western blot and other molecular biology methods, the subject studied ginsenoside Rb1 which influence FFAs spillover from fat cells at basis or the insulin resistance state, clarifies antidiabetic mechanism of Rbl.Objective:Studying the mechanism of ginsenoside Rbl to free fatty acid overflow from fat cells by observing the effect of ginsenoside Rb1 on 3T3-L1 adipocyte in vitro and in obese mice induced by Fat diet in vivo.Methods:(1)Observing Ginsenoside Rbl that influences Free fatty acid overflow of 3T3-L1 adipocyte in basis and insulin resistance state. (2) Studying the expression of Perilipin in insulin resistance state 3T3-L1 adipocyte by Western blot. (3)Researching the effect of ginsenoside Rb1 on Perilipin protein translocation in normal state in adipocyte using laser scanning confocal microscope. (4)Researching the effect of ginsenoside Rb1 on the epididymal fat content/body weight,NEFA, TC, TG, HDL-C, LDL-C,TNF-a, IL-6 in obese mice.Results:(1) The study show that Ginsenoside Rb1 neither have significant inhibition on basal 3T3-L1 adipocyte lipolysis, nor remarkable increment of lipolysis caused by TNF-a in 3T3-L1 adipocyte in insulin resistance state; (2) TNF-a induced insulin resistance state in 3T3-L1 adipocyte, brought about Perilipin expression decreased significantly. After the intervention of Ginsenoside Rbl for 24 hours, it reversed down-regulated expression of Perilipin caused by TNF-a in 3T3-L1 adipocyte; (3) Rb1 significantly increased the Perilipin protein translocation from cytoplasm to lipid droplets surface in normal state in 3T3-L1 adipocyte. (4) Ginsenoside Rb1 significantly reduced the epididymal fat content/body weight in obese mice; ginsenoside Rbl had no significant effect on NEFA, TC, TG, HDL-C, LDL-C levels; ginsenoside Rb1 can improve insulin resistance in obese mice and decrease TNF-a, IL-6 level of obese mice; ginsenoside Rbl upregulated the phosphorylation level of HSL,had no significant effect on the expression of HSL in epididymal fat in obese mice.Conclusion:(1) Ginsenoside Rbl may inhibit adipocyte lipolysis in normal state in 3T3-L1 adipocyte to a certain extent; (2) Ginsenoside Rbl may inhibit the lipolysis level increasement caused by TNF-a in 3T3-L1 adipocyte; (3) Ginsenoside Rbl may inhibit lipolysis under basal condition by promoting fat cells translocation of Perilipin from cytoplasm to Lipid droplet surface; may be passed against TNF-a expression caused by the reduction in Perilipin inhibited lipolysis insulin resistance; (4) Ginsenoside Rbl may reduce NEFA levels, fat in obese mice, improving lipid metabolism, improved insulin resistance in obese mice, inhibiting TNF-a, IL-6 and other inflammatory cytokines.ginsenoside Rb1 inhibited lipolysis by upregulating the phosphorylation level of HSL更多还原