【作者】 董劭壮；

【导师】 赵水平；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 目的肿瘤坏死因子α(TNF-α)作为促炎因子对脂肪细胞功能有多方面的影响，包括促进细胞分化、脂解及脂肪因子分泌等。本文拟研究TNF-α对传代培养的兔皮下脂肪细胞胆固醇流出功能的影响，及可能的作用机制。方法来源于兔腹股沟皮下的脂肪细胞以不同浓度的TNF-α(5，10，20 ng／ml)干预24小时。用液体闪烁计数仪测量脂肪细胞对氚标胆固醇的流出率。用逆转录多聚酶链式反应(RT-PCR)检测过氧化物酶体增殖物激活受体γ(PPARγ)、肝X受体α(LXRα)及三磷酸腺苷结合盒转运体A1(ABCA1)的mRNA表达。结果1．5或10 ng／ml TNF-α干预脂肪细胞24小时，可使胆固醇流出率增加，且10 ng／ml TNF-α作用最强(7.03±1.17％)，较对照组(3.92±0.46％)明显增高。但20 ng／ml TNF-α反而使胆固醇流出率较10 ng／ml时下降了16.2％(P＜0.05)。2．5或10 ng／ml TNF-α可上调ABCA1表达，其表达量分别为0.44±0.17和0.91±0.26，与对照组0.20±0.06相比差异有显著性。20 ng／ml TNF-α使ABCA1表达有所下降(0.69±0.13)。与此同时，TNF-α可调节脂肪细胞PPARγ和LXRα表达。在TNF-α≤10 ng／ml时起促进作用，在20 ng／ml时起抑制作用。3．预先以PPARγ特异性抑制剂GW9662孵育半小时，可抑制10 ng／ml TNF-α对PPARγ(0.88±0.22 vs 1.37±0.25，P＜0.05)和LXRα(0.51±0.14 vs 0.32±0.08，P＜0.05)的诱导作用。结论TNF-α影响脂肪细胞胆固醇流出及其ABCA1表达，这种作用与TNF-α对脂肪细胞PPARγ-LXRα的双重调节作用有关。目的脂肪细胞肥大伴随一系列功能改变。我们拟研究在氧化低密度脂蛋白(oxLDL)诱导脂肪细胞负荷脂质后，TNF-α对其胆固醇流出和ABCA1表达的影响是否会发生改变。方法用50μg／ml oxLDL孵育脂肪细胞24小时制造荷脂模型。再以不同浓度TNF-α(5，10，20 ng／ml)干预24小时，观察胆固醇流出变化及用RT-PCR法检测脂肪细胞PPARγ、LXRα和ABCA1 mRNA表达。结果1．OxLDL可使脂肪细胞胆固醇流出增加(7.46±2.34％vs 3.92±0.46％)，PPARγ(1.07±0.26 vs 0.48±0.12)、LXRα(0.83±0.16vs 0.32±0.08)和ABCA1(1.19±0.32 vs 0.20±0.06)表达上升，P均＜0.05。2．TNF-α抑制荷脂脂肪细胞胆固醇流出，10 ng／ml时作用最强(6.02±0.31％vs3.92±0.46％)，在20 ng／ml时脂肪细胞胆固醇流出率略有回升(6.12±0.53)，差异有显著性。3．从5 ng／ml到20 ng／ml TNF-α，PPARγ表达量分别为0.93±0.21，0.77±0.15，0.60±0.16与oxLDL对照组1.07±0.26相比表达降低。LXRα表达量为0.72±0.19，0.61±0.17，0.49±0.09与oxLDL对照组0.83±0.16相比也有降低。TNF-α呈浓度依赖性抑制脂肪细胞PPARγ和LXRα表达。5 ng／ml和10 ng／ml TNF-α分别使脂肪细胞ABCA1表达下降了14.4％和34.5％，20ng／ml TNF-α使ABCA1表达有所回升。结论在oxLDL诱导荷脂的脂肪细胞中，TNF-α对PPARγ和LXRα表达主要表现为抑制作用，并在一定程度上影响到ABCA1表达及脂肪细胞胆固醇流出。目的姜黄素是一类中药提取物，已证实其具有抗炎、抗氧化及改善血脂异常等作用。本研究观察了姜黄素对脂肪细胞抗炎脂肪因子脂联素表达和分泌的影响，初步评价姜黄素对脂肪细胞功能的影响。方法新西兰大白兔腹股沟皮下脂肪细胞以不同浓度姜黄素(5，10，20μg／ml)孵育24小时，用ELISA试剂盒检测细胞培养上清液脂联素水平，并用RT-PCR方法检测脂联素及其上游因子PPARγ的表达。结果5μg／ml至20μg／ml姜黄素分别使兔皮下脂肪细胞脂联素分泌水平较对照组(3.13±0.21μmol／l)增加(3.93±0.26，4.49±0.34，5.21±0.39μmol／l)。脂联素mRNA表达也较对照组(0.40±0.10)增加(0.55±0.09，0.66±0.11，0.80±0.13)。姜黄素促进PPARγ表达的作用与脂联素表达的改变一致，并在20μg／ml时达到最大(1.01±0.15vs 0.48±0.12，P＜0.05)。结论姜黄素可促进兔皮下脂肪细胞脂联素的表达和分泌，这种作用与其上调脂肪细胞PPARγ表达有关。更多还原

【Abstract】 ObjectiveThe pro-inflammatory cytokine TNF-αhas multiple effects on adipocyte function, including differentiation, lipolyis and the production of adipokines. In this paper, we have evaluated the effects of TNF-αon cholesterol efflux in passaged rabbit subcutaneous adipocyte, which had been elucidated in macrophages, and the possible mechanism involved.MethodsPassaged rabbit inguina subcutaneous adipocytes were incubated with 5, 10, 20 ng/ml different doses of TNF-αfor 24 hrs. The cholesterol effiux onto apolipoprotein AⅠ(apoAⅠ) was assessed, and the related peroxisome proliferator activated receptorγ(PPARγ), liver X receptorα(LXRα) and ATP-binding cassette transporter A1 (ABCA1) mRNA expression in adipocytes were quantitated by reverse transcription polymerase chain reaction (RT-PCR).Results1. Treatment of normal passaged adipocytes with TNF-α(5 or 10 ng/ml) for 24 hrs increased the level of cholesterol effiux onto apoAⅠ, and the effct of 10 ng/ml TNF-αwas significant (7.03±1.17% vs 3.92± 0.46%, P＜0.01). In contrast, 20 ng/ml TNF-αdecreased cholesterol effulux by 16.2% compared with 10 ng/ml TNF-α( P<0.05).2. The expression of ABCA1 was elevated under treatment of 5 or 10 ng/ml TNF-α(0.444±0.17 vs 0.204±0.06, 0.914±0.26 vs 0.20±0.06, respectively, P＜0.05), and inhibited by 20 ng/ml TNF-α(0.694±0.13). In consistence with the change of ABCA1, the PPARγand LXRαmRNA were significantly induced by 10 ng/ml TNF-α(1.36±0.25 vs 0.48±0.12, 0.844±0.18 vs 0.324±0.08) and downregulated by higher TNF-α.3. After pre-treated by PPARγspecial inbhibitor GW9662 for half an hour, the introduction of PPARγby 10 ng/ml TNF-αwas partially prevented, subsequent to the downregulation of LXRαand ABCA1.ConclusionTNF-αaffects cholesterol effiux and ABCA1 expression of adipocytes, and the pathway of PPARγ-LXRα-ABCA1 is probably involved. ObjectiveObesity is caused by increased adipose mass, which is characterized with enlarged adipocytes. The energy homeostasis management and endocrinal function of enlarged fat cell under certain state, e.g lipid-laden, will be different with that of normal cells. Here we have investigated the effect of pro-inflammatory cytokine TNF-αon cholesterol effulux in the cholesterol laden adipocytes, which was induced by oxidized low density lipoprotein (oxLDL), and the possible mediators in its action.MethodsAdipocyte isolated from the New Zealand White Rabbit inguina subcutaneous adipose were incubated with 50/zg/ml oxLDL for 24 hrs, then were treated with different doses of TNF-α(5, 10, 20 ng/ml) for another 24 hrs. The cholesterol efflux onto apoAⅠwas assessed. The related gene of PPARγ, LXRαand ABCA1 expression in adipocytes were evaluated by RT-PCR.Results1. Cholesterol efflux was induced by uptake of oxLDL in adipocyte (7.46±2.34% vs 3.92±0.46%, P＜0.05). TNF-αsignificantly inhibited the effects of oxLDL under 5 or 10 ng/ml (6.02±0.31%, 4.56±1.62%). 20 ng/ml TNF-αreversed the downregulation to some extent (6.12±0.53%).2. ABCA1 mRNA was increased in lipid-laden adipocyte induced by oxLDL (1.19±0.32 vs 0.20±0.06), and decreased under addition of 5 ng/ml TNF-α(1.02±0.29) or 10 ng/ml TNF-α(0.78±0.24). In contast, 20 ng/ml TNF-αprevented the downregulation (0.98±0.17).3. The induction of PPARγand LXRαmRNA in lipid-laden adipocyte were decreased by TNF-αin a dose dependent pattern. From the low to the high dose of TNF-α, PPARγexpression was 0.93±0.21, 0.77±0.15, 0.60±0.16 compared with oxLDL control group 1.07±0.26. LXRαexpression was 0.72±0.19, 0.61±0.17, 0.49±0.09 compared with oxLDL control group 0.83±0.16.ConclusionTNF-αdownregulated the augmentation of PPARγand LXRαresulted from lipid burden in rabbit subcutaneous adipocyte induced by oxLDL, and subsequently influenced the expression of ABCA1 and cholesterol effiux. ObjectiveTo investigate the potential benefits of curcumin, an extract from traditional medical plants, on adipocyte function.MethodsRabbit subcutaneous adipocytes were treated with 5, 10, 20μg/ml curcumin for 24 hrs. One of the major adipokines, adiponectin secretion in supernatant was examined by special ELISA kit. The expression of adiponectin and PPARγ, its upstream regulator, were quantitated by RT-PCR.Results1. Curcumin increased the adiponectin secretion examined in supernatant in a dose dependent pattern. From 5μg/ml to 20μg/ml, the secretion of adiponectin were 3.93±0.26, 4.49±0.34, 5.21±0.39 vs 3.13±0.21μmol/1 (control group).2. Curcumin induced the expression of PPARγin cultured adipocyte, and reached its peak value on 20μg/ml (1.01±0.15 vs 0.48±0.12). The adiponectin mRNA was also increased under treatment of all the doses of curcumin (0.55±0.09, 0.66±0.11, 0.80±0.13 vs 0.40±0.10).ConclusionCurcumin is able to induce the expression and secretion of adiponectin in adipocyte, which related to its upregulation of expression of PPARγ.更多还原