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背景:瘦素是脂肪组织分泌的一种多肽激素,研究显示瘦素在动脉粥样硬化形成中发挥了一定重要的作用。目的:观察瘦素对鼠源性巨噬细胞系RAW264.7细胞肿瘤坏死因子α表达的影响,并从核转录因子κB活性变化角度探讨其可能机制。设计:对照观察实验。单位:华中科技大学同济医学院生物化学及分子生物学系。材料:实验于2005-04/2006-02在华中科技大学同济医学院生物化学及分子生物学系及附属协和医院普外科实验室完成。将培养的RAW264.7细胞分为不同浓度瘦素处理组(12.5,25,50,100μg/L)、IkappaB激酶抑制剂组及空白对照组。每组3瓶,重复实验3次。方法:将鼠源性巨噬细胞株RAW264.7细胞以1×109L-1密度接种于6孔板中,用含体积分数为0.1的小牛血清的RPMI-1640培养基培养。待RAW264.7细胞生长至80%时,换用无血清培养基Opti-MEM继续培养24h后,将细胞分为不同浓度瘦素处理组(12.5,25,50,100μg/L)及空白对照组,瘦素孵育4h后采用反转录-聚合酶链反应检测肿瘤坏死因子α在mRNA水平的表达。上述分组细胞经瘦素分别孵育1,3,6和9h后采用双抗夹心酶联免疫吸附实验检测肿瘤坏死因子α在蛋白水平的表达。上述分组细胞经瘦素孵育不同时间后采用凝胶迁移率实验检测细胞核内核转录因子κB活性。将RAW264.7细胞分为以下4组:空白对照组、IkappaB激酶特异性抑制剂PS1145(10μmol/L)处理组、瘦素(50μg/L)处理组、瘦素(50μg/L)+PS1145(10μmol/L)组,各组孵育时间均为6h,分别检测细胞核内核转录因子κB活性及肿瘤坏死因子α在mRNA水平的表达。主要观察指标:①不同浓度瘦素对RAW264.7细胞肿瘤坏死因子α:mRNA表达水平的影响;蛋白分泌的影响。②不同浓度瘦素对RAW264.7细胞核内核转录因子κB活性的影响。③抑制IkappaB激酶活性对瘦素诱导RAW264.7细胞肿瘤坏死因子α的影响。结果:①RAW264.7细胞经不同浓度的瘦素处理后,肿瘤坏死因子α在mRNA水平呈瘦素剂量依赖性增加,50μg/L瘦素处理组达峰值。②蛋白水平的表达呈瘦素剂量时间依赖性增加,50μg/L瘦素处理6h即可达峰值。③核转录因子κB的活性亦与瘦素浓度正相关,50μg/L瘦素处理6h后核转录因子κB活性最高(P<0.05)。④抑制IkappaB激酶活性可部分抑制肿瘤坏死因子α的表达。结论:瘦素可直接促进RAW264.7细胞肿瘤坏死因子α的表达和分泌,并呈剂量时间依赖性,其机制可能与瘦素激活核转录因子κB有关。这可能是瘦素致动脉粥样硬化的机制之一。
BACKGROUND: Leptin is a polypeptide hormone secreted by adipose tissue. Studies have shown that leptin plays an important role in the development of atherosclerosis. OBJECTIVE: To observe the effect of leptin on tumor necrosis factor-α (TNF-α) expression in murine macrophage RAW264.7 cells and to explore its possible mechanism from the point of view of the change of nuclear factor kappa B activity. Design: Control observation experiment. SETTING: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: The experiment was performed at Department of General Surgery, Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology and affiliated Union Hospital from April 2005 to February 2006. The cultured RAW264.7 cells were divided into different concentrations of leptin group (12.5,25,50,100μg / L), IkappaB kinase inhibitor group and blank control group. 3 bottles in each group, repeat experiment 3 times. Methods: Murine macrophage RAW264.7 cells were seeded into 6-well plates at a density of 1 × 109L-1 and cultured in RPMI-1640 medium containing 0.1% fetal bovine serum. When RAW264.7 cells were grown to 80%, the cells were further cultured in serum-free medium Opti-MEM for 24 hours and then divided into different concentrations of leptin group (12.5, 25, 50, 100μg / L) and blank control group, After 4 h incubation with leptin, the expression of tumor necrosis factor alpha (TNF-α) at the mRNA level was detected by reverse transcription-polymerase chain reaction. The cells were incubated with leptin for 1, 3, 6 and 9 h, respectively. The levels of tumor necrosis factor alpha (TNF-α) at the protein level were detected by double-antibody sandwich enzyme-linked immunosorbent assay. The above group of cells were incubated with leptin for different time and the activity of nuclear factor kappa B (NF-κB) in the nucleus was detected by gel shift assay. RAW264.7 cells were divided into the following 4 groups: blank control group, IkappaB kinase-specific inhibitor PS1145 (10μmol / L), leptin (50μg / L) 10μmol / L). The incubation time of each group was 6h. The activity of nuclear factor κB and the expression of tumor necrosis factor α in the nucleus were detected. MAIN OUTCOME MEASURES: ① Effects of different concentrations of leptin on the expression of tumor necrosis factor α: mRNA in RAW264.7 cells and the effect of protein secretion. Effects of leptin at different concentrations on the activity of nuclear factor kappa B in RAW264.7 cells. ③ Inhibition of IkappaB kinase activity on leptin-induced tumor necrosis factor-α in RAW264.7 cells. Results: ①RAW264.7 cells treated with different concentrations of leptin showed a dose-dependent increase in tumor necrosis factor α at the mRNA level and a peak at 50 μg / L leptin treatment group. ② The protein level was increased in a dose-dependent manner by leptin, and reached the peak at 6h after treatment with 50μg / L leptin. The activity of nuclear factor kappa B was also positively correlated with leptin concentration. The activity of nuclear factor kappa B was the highest at 6h after treatment with 50μg / L leptin (P <0.05). ④ Inhibition of IkappaB kinase activity can partially inhibit the expression of tumor necrosis factor-α. Conclusion: Leptin can directly promote the expression and secretion of tumor necrosis factor - α in RAW264.7 cells in a dose - and time - dependent manner, which may be related to the activation of nuclear factor κB by leptin. This may be one of the mechanisms by which leptin causes atherosclerosis.