【作者】 姚伊人；

【导师】 叶菜英； 张德昌；

【作者基本信息】 中国协和医科大学 ， 药理学， 2009， 硕士

【摘要】 研究目的:羧胺三唑（Carboxyamido-triazole,CAI）是一个主要由美国国家癌症中心研究的非细胞毒类抗肿瘤新药,在美国已进行过若干实验室和临床研究,其抗肿瘤特性已经得到证实。我们实验室发现羧胺三唑的抗癌作用可能与其细胞周期特异的抑制作用有关,同时发现其具有抗炎作用。本文将进一步证明其抗炎作用,同时以巨噬细胞为对象,研究其抗炎机理,并希望找到羧胺三唑在抗炎和抗肿瘤作用机理中的共同点。研究内容:1.建立大鼠佐剂性关节炎模型,检测CAI对在该慢性炎症模型上的抗炎表现。2.以体外培养的大鼠肺泡巨噬细胞系（NR8383）为对象,用脂多糖（LPS）诱导建立炎症模型,检测CAI对其上清中TNF-α,IL-1β和NO的影响,并进一步检测相关信号通路中典型蛋白含量的变化。研究方法:本文采用大鼠佐剂性关节炎模型来检测羧胺三唑对慢性炎症和免疫性炎症的作用;采用体外培养的大鼠肺泡巨噬细胞系NR8383经LPS刺激后,加入CAI(10μmol.L⁽-1(,401×mol.L^-1)用酶联免疫吸附法（ELISA）检测细胞上清中TNF-α、NO和IL-1β的水平,用Western blot检测细胞裂解物中IκBα,磷酸化IκBα,和磷酸化JNK蛋白含量的变化。研究结果:1.在0.1 ml完全弗氏佐剂诱导的佐剂性关节炎模型中,20 mg/kg羧胺三唑可以抑制佐剂性关节炎大鼠的原发病变足和继发病变足的足趾肿胀度,与PEG400溶剂对照组相比,有显著差异（P＜0.05,P＜0.01）。2.10和40μmol·L^-1的羧胺三唑都能降低LPS刺激的NR8383细胞系上清中一氧化氮的含量,使NO含量从33.63±0.801amol·L^-1分别降至5.11±0和4.43±0.32μmol·L^-1,抑制率分别达到84.8%和86.8%,与DMSO溶剂对照组相比,有显著差异（P＜0.01）。3.40μmol·L^-1的羧胺三唑能降低LPS刺激的NR8383细胞系上清中TNF-α的含量,使TNF-α含量从224.03±8.94 pg·ml^-1降至185.5±8.53 pg·ml^-1,抑制率达到17.2%,与DMSO溶剂对照组相比,有显著差异（P＜0.05）。4.10和40μmol·L^-1的羧胺三唑都能降低LPS刺激的NR8383细胞系上清中IL-1β的含量,使IL-1β含量从31.97±3.14 pg·ml^-1分别降至13.5±0.98和8.43±0.34pg·ml^-1,抑制率分别达到57.8%和73.6%,与DMSO溶剂对照组相比,有显著差异（P＜0.05,P＜0.01）。5.10和40μmol·L^-1的羧胺三唑对LPS刺激的NR8383细胞系中IκBα,磷酸化IκBα,和磷酸化JNK蛋白含量没有明显影响。结论:羧胺三唑有抗炎作用,其作用机制可能与抑制巨噬细胞分泌TNF-α、IL-1β和NO等炎症因子有关。但该抑制作用可能不是通过抑制NF-κB和JNK信号通路的激活来实现的。更多还原

【Abstract】 OBJECTIVE:Carboxyamido-triazole（CAI） is an experimental anti-cancer agent developed by National Institute of Cancer（NCI） of National Institutes of Health（NIH） of the United States of American.The anti-cancer effects of CAI have been proved by many preclinical experiments and several clinical trails in different stages carried out in the United States.We have found the mechanism of CAI’s anti-tumor effect may be associated with inhibition of cell cycle.Meanwhile,we found CAI has a effect of anti-inflammatory.In this paper,we will prove the anti-inflammatory effect of CAI,and research the mechanism in macrophage.Base on these datas we hope to find the same target between anti-tumor and anti-inflammatory effects.Contents:1.Evaluate the effect of CAI on chronic inflammation and immunity inflammation in adjuvant-induced arthritis rat model.2.Use the model of lipopolysaccharide（LPS） stimulated rat alveolar macrophage cell line（NRg383）,test the inhibition effect of CAI on TNF-α,IL-1βand NO.And then observe the different in cell signal pathway.Methods:This study adopted adjuvant-induced arthritis rat model to evaluate the effect of CAI on chronic inflammation and immunity inflammation;this study cultured NR8383 cells were stimulated by LPS,added CAI,then use enzyme-linked immunosorbent assay （ELISA） to detect cell supematant TNF-α,NO and IL-1βlevels,and Western blot to detect celt lysates of IκBα,phosphorylated IκBα,and phosphorylated JNK content changes. Results:1.CAI inhibited the primary lesions and secondary lesions of AIA rats at 20 mg/kg,significantly（compare with PEG400 control group,P＜0.05,P＜0.01）.2.CAI at 10 and 40μmol·L^-1 both decreased the levels of NO in supematant of NR8383 cell line stimulated by LPS.The level of NO for the DMSO control group was found to be 33.63±0.80μmol·L^-1.Treatment with CAI at 10 and 40μmol·L^-1 decreased the levels to 5.11±0 and 4.43±0.32μmol·L^-1,respectively.The inhibitory rate are 84.8% and 86.8%,respectively.（P＜0.01）3.CAI at 40μmol·L^-1 both decreased the levels of TNF-αin supernatant of NR8383 cell line stimulated by LPS.The level of TNF-αfor the DMSO control group was found to be 224.03±8.94 pg·ml^-1.Treatment with CAI at 40μmol·L^-1 decreased the levels to 185.5±8.53 pg·ml^-1.The inhibitory rate is 17.2%.（P＜0.05）4.CAI at 10 and 40μmol·L^-1 both decreased the levels of IL-1βin supernatant of NR8383 cell line stimulated by LPS.The level of IL-1βfor the DMSO control group was found to be 31.97±3.14 pg·ml^-1.Treatment with CAI at 10 and 40μmol·L^-1 decreased the levels to 13.5±0.9 and 8.43±0.34 pg·ml^-1,respectively.The inhibitory rate are 57.8 and 73.6%,respectively.（P＜0.05,P＜0.01）5.CAI at 10 and 40μmol·L^-1 have no significant effect on IκBα,phosphorylated IκBα, and phosphorylated JNK in which NR8383 cell line stimulated by LPS.Conclusion:CAI can probably play an anti-inflammatory effect via inhibiting cytokines NO,IL-1βand TNF-α,but not acting on the NF-κβand JNK such classical signaling pathways.更多还原