【摘要】 目的:肥大细胞(MCs)类胰蛋白酶是一种丝氨酸蛋白酶,目前未发现其体内内生的抑制剂。本研究用水蛭源性类胰蛋白酶抑制剂(LDTI)研究MCs颗粒释放液抑制THP-1巨噬细胞源性泡沫细胞胆固醇流出的机制。方法:用Compound 48/80刺激大鼠腹腔MCs活化,制备颗粒释放液。荧光分光光度仪检测MCs组胺释放率。采用丙酮抽提LDTI并用反相高效液相纯化。酶法检测MCs颗粒释放液中类胰蛋白酶的活性。用15%非还原聚丙烯酰胺凝胶电泳检测HDL3的改变。用高效液相色谱检测细胞内总胆固醇(TC)、游离胆固醇(FC)和胆固醇酯(CE)含量,液体闪烁计数器检测细胞内胆固醇流出。结果:MCs颗粒释放液使泡沫细胞内胆固醇流出减少,总、游离胆固醇蓄积增加,HDL3的载脂蛋白A-Ⅰ降解;而用LDTI抑制MCs颗粒释放液中类胰蛋白酶的活性后,可取消MCs颗粒释放液的这些作用。结论:LDTI能抑制MCs颗粒释放液的作用,表明类胰蛋白酶可以抑制胆固醇外流,而且与HDL3的apoA-Ⅰ的降解有关。更多还原

【Abstract】 Objective Mast cells（MCs） tryptase is a kind of serine protease,and no endogenous inhibitors have been described for it.In the present study,we investigate the mechanism of MCs-inhibited cholesterol efflux from THP-1 macrophage-derived foam cells by leech-derived tryptase inhibitor（LDTI）.Methods MCs,isolated from peritoneal cavities of rats,were stimulated to degranulate with Compound 48/80.The degree of MCs degranulation was determined by measuring the histamine release percentage using fluorescence spectrometer.LDTI was extracted by acetone and purified by reverse-phase high performance liquid chromatography（HPLC）.Tryptase activity in MC granules was assayed by enzymology.HDL₃that was treated with MC granules and LDTI was analyzed by 15% SDS-PAGE under nonreducing conditions.The level of intracellular cholesterol and cholesterol efflux were determined by HPLC,and FJ-2107P type liquid scintillator,respectively.Results Incubation of the MC granules with THP-1 macrophage-derived foam cells and HDL₃ led to decrease of high-affinity efflux of cholesterol from THP-1 macrophage-derived foam cell,increase of total,free cholesterol level.Treatment of HDL₃ with the MC granules resulted in rapid degradation of the main HDL₃apoliproteins,apoA-Ⅰ.However,after LDTI had inhibited the tryptase activity,the above effects of MC granules were attenuated.Conclusion LDTI impairs ability of MC granules to inhibit cholesterol efflux,which indicates tryptase may restrain cholesterol efflux by degrading apoA-Ⅰof HDL₃.更多还原