【作者】 蒋佩；

【导师】 廖端芳； 严鹏科；

【作者基本信息】 南华大学 ， 药理学， 2005， 硕士

【摘要】 目的:研究胆固醇流出对ox-LDL诱导的巨噬细胞凋亡的影响。 方法:用50mg/Lox-LDL与RAW264.7细胞共同孵育0,12,24,48,96小时。用50mg/Lox-LDL处理RAW264.7细胞48小时后加入不同浓度的HDL₃（50,100,200mg/L）和胆固醇流出抑制剂Brefeldin（BFA）或胆固醇流出促进剂β-cyclodextrin（β-CD）孵育24小时。油红O染色观察细胞内脂滴的形成情况;高效液相分析法检测细胞内胆固醇含量;液体闪烁计数器检测细胞内胆固醇流出;Hoechst33258观察细胞凋亡形态;流式细胞仪检测细胞凋亡。 结果:用50mg/Lox-LDL与RAW264.7细胞共同孵育0,12,24,48,96小时,结果显示:RAW264.7细胞的凋亡率随着处理时间的延长而显著提高。在50mg/Lox-LDL与RAW264.7细胞共同孵育48小时出现明显的凋亡形态学改变（胞质体积缩小,胞核凝集、边缘化）,油红O染色显示,细胞内的大量脂滴形成,细胞胆固醇酯含量显著增加,细胞呈典型泡沫化形态。用50mg/Lox-LDL处理RAW264.7细胞48小时后,加入不同浓度的HDL₃（50,100,200mg/L）和胆固醇流出抑制剂BFA（4μmol）或β-CD（10mmol/L）孵育24小时。结果显示:HDL₃呈浓度依赖性的促进细胞内胆固醇流出和降低细胞内的胆固醇含量,油红O染色显示,细胞内的脂滴明显减少。细胞的凋亡率也随着HDL₃浓度的升高而逐渐降更多还原

【Abstract】 AIM: To explore the effect of cholesterol efflux on the oxidized low density lipoprotein (ox-LDL)-induced apoptosis in macrophages.METHODS: The concentration of cellular cholesterol was detected by highperformance liquid chromatography; Oil Red O Dyeing experiment was used to show the cellular lipid droplets in cells; Cholesterol efflux was determined by FJ-2107P type liquid scintillator; Apoptosis cells were confirmed by Hoechst 33258 staining and flow cytometric analysis.RESULTS: After treated with 50mg/L ox-LDL for 0, 12, 24, 48, 96h, the apoptoticrate of RAW264.7 macrophages increased in a time-dependent manner. Typical morphological change of apoptosis such as condensed chromatin and shrunken nucleus markedly increased. When cells were treated with ox-LDL for 48h, many lipid droplets accumulated in cells, and the content of cellular total cholesterol and cholesterol ester also increased obviously. Foamy cells induced by ox-LDL, were then treated with various concentration of HDL3 (50,100,200mg/L) for 24h, the rate of intracellular cholesterol efflux increased obviously and the concentration of cellular cholesterol decreased in a dose-dependent manner. Meanwhile, the apoptotic rate decreased in a dose-dependent manner, typical morphological change of apoptosis also descended. However, Brefeldin(BFA), an inhibitor of cholesterol efflux, markedly attenuated the protective effect of HDL3 on ox-LDL-induced apoptosis, and increased the content of total cholesterol, lipid droplets in cells when compared with the group of HDL3 (200mg/L) alone. Meantime, 10mmol/L β-cyclodextrin (β-CD), a更多还原