【作者】 赵战芝；

【导师】 杨永宗；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2005， 硕士

【摘要】 目的 肥大细胞是一种参与炎症免疫应答反应的细胞。长期以来人们对肥大细胞的研究都侧重于过敏、皮炎、哮喘、关节炎等炎性及免疫性疾病。随着近年来人们对动脉粥样硬化的认识不断深入,从单纯的脂质学说到炎症学说,发现肥大细胞与动脉粥样硬化的发生发展密切相关。本研究以THP-1巨噬细胞源性泡沫细胞为研究对象,以药用水蛭源性类胰蛋白酶抑制剂为干预试剂,研究肥大细胞（肥大细胞颗粒释放液）对高密度脂蛋白3介导其胆固醇流出的影响,并初步探讨其作用机制。 方法 用Compound 48/80处理大鼠腹腔肥大细胞后,采用荧光分光光度仪检测组胺释放率,甲苯胺蓝染色观察肥大细胞脱颗粒状态。LDTI的提取采用丙酮抽提,反相高效液相纯化。酶法检测MCs颗粒释放液中类胰蛋白酶的活性。THP-1巨噬细胞源性泡沫细胞在有或无HDL₃、肥大细胞颗粒释放液、LDTI的培养基里共育20h,油红O染色检测泡沫细胞内脂质,高效液相色谱检测细胞内胆固醇和CE,液体闪烁计数器、荧光分光光度仪检测细胞内胆固醇的流出;RT-PCR检测THP-1巨噬细胞源性泡沫细胞三磷酸腺苷结合盒转运体A1mRNA的转录水平。15%非还原聚丙烯酰胺凝胶电泳、琼脂糖凝胶电泳检测MCs颗粒释放液处理后的HDL₃。 结果 肥大细胞被Compound 48/80处理后,甲苯胺蓝染色显示肥大细胞明显脱颗粒,组胺释放率为81.53±4.51%,表明肥大细胞脱颗粒度达到更多还原

【Abstract】 OBJECTIVE: Mast cells (MCs) are better known for their actions in inflammationallergic reactions. Traditionally interest has centered around the role of MCs in inflammation or allergic disease such as hypersusceptibility, dermatitis, asthma, arthritis. Having understood the atherosclerosis, researchers discover that MCs might be associated with the pathogenesis of atherosclerosis. In human atherosclerotic lesions, degranulated mast cells are found to be present in the close vicinity of cholesterol-loaded macrophage-derived foam cells. In the present study, we investigate whether the MCs caninhibit HDL3 -induced cholesterol efflux from THP-1 macrophage-derived foam cells and its possible reasons.METHODS: MCs, isolated from peritoneal cavities of rats, were stimulated todegranulate with Compound 48/80. The degree of MCs degranulation was determined by measuring the histamine release percentage using luminescence spectrometer. The morphology characters of MCs was detected by microscope, after toluidine blue staining. LDTI were extracted by acetone and purified by reverse-phase HPLC. Tryptase activities in supernatant of MCs were assayed by reacting with substrate BAPNA.After THP-1 macrophage-derived foam cells were incubated in the absence or presence of mast cells supernatant 、leech-derived tryptase inhibitor(LDTI) and HDL3 in 20 hours, the intracellular lipid droplets was observed with oil red O staining, the level of intracellular cholesterol and cholesterol efflux were determined by high performance liquid chromatography (HPLC ) ,and FJ-2107P type liquid scintillater、 luminescence spectrometer, respectively. The mRNA expression of ABCA1 was examined by RT-PCR. HDL3 that was treaded with supernatant of mast cells was analyzed by 15% SDS-PAGE under nonreducing conditions and agarose gel electrophoresis.更多还原