【作者】 孙少卫；

【导师】 廖端芳；

【作者基本信息】 南华大学 ， 药理学， 2007， 硕士

【摘要】 目的:研究氧化低密度脂蛋白跨内皮细胞转运的方式,试图揭示caveolae在胆固醇流出中的作用机制,并探讨caveolin-1与内皮细胞胆固醇摄取间存在的关系,寻找是否存在调节关系。方法:用40μg/ml氧化低密度脂蛋白处理人脐静脉内皮细胞不同时间,分别用荧光和HPLC观察细胞内胆固醇含量变化;随后将已用氧化低密度脂蛋白荷脂的人脐静脉内皮细胞,撤去氧化低密度脂蛋白后再观察细胞内胆固醇含量变化。分别用三种caveolae阻滞剂carrageenan、filipin、nocodazole预处理人脐静脉内皮细胞1h后与40μg/ml氧化低密度脂蛋白孵育24h,用荧光倒置显微镜观察内皮细胞荷脂情况。将人脐静脉内皮细胞种植于transwell培养系统套皿上层,分别用三种阻滞剂预处理细胞1h后加入40μg/mlDiI荧光标记的氧化低密度脂蛋白于套皿上层孵育24h,取套皿下层液体用荧光分光光度计测荧光值。用FITC标记LOX-1后,用nocodazole阻滞cavolae运动,用荧光倒置显微镜观察LOX-1的运动情况。利用caveolin-1 SiRNA干扰技术沉默内皮细胞caveolin-1表达,同时检测内皮细胞在氧化低密度脂蛋白作用下NF-κB和LOX-1的表达情况。结果:实验表明人脐静脉内皮细胞与氧化低密度脂蛋白孵育时,细胞荷脂呈时间依赖性增加,撤去氧化低密度脂蛋白后细胞内脂质逐渐减少。caveolae阻滞剂可明显阻滞内皮细胞对ox-LDL摄取,同时也可明显阻滞氧化低密度脂蛋白跨内皮细胞转运达49%, 72%和80%。免疫荧光结果表明LOX-1定位于caveolae中。SiRNA干扰实验表明沉默caveolin-1表达后,抑制了NF-κB核转位,同时LOX-1表达下调。结论:1.内皮细胞可摄取ox-LDL而荷脂,也可自发性流出所荷脂质。内皮细胞的ox-LDL受体-LOX-1-定位于caveolae中,caveolae在内皮细胞摄取ox-LDL的过程中起了一个运载体的作用,它同时也介导了ox-LDL的跨内皮细胞转运。2. caveolin-1促使NF-κB核转位而活化,进一步上调LOX-1表达。更多还原

【Abstract】 Objective: To explore the mechanisms involved in cholesterol transcytosis across endothelial cells and the effects of caveolae in this process,and determine the relation between caveolin-1 expression and cholesterol efflux in human umbilical vein endothelial cellsMethod: Human umbilical vein Endothelial Cells(HUVEC) were incubated with ox-LDL(40μg/ml) for different times,and depleted ox-LDL from medium after lipid-loaded 24h for different times, we used fluorescence microscope and HPLC to determine the cholesterol content in HUVEC at varies times. We first incubated HUVEC with carrageenan, filipin, or nocodazole for 1h, and a 24h incubation was followed after addition of DiI-ox-LDL(40μg/ml), the DiI fluorescent was observed with fluorescent microscope after incubation. We grown HUVEC on a Transwell insert to obtain a tight monolayer and preincubated with carrageenan, filipin, or nocodazole in the apical reservoir for 1h, then incubated 24h after addition of DiI-ox-LDL(40μg/ml), at the end of incubation period, the fluorescence intensity of DiI-ox-LDL that transcytosised up to the bottom reservoir was determined by Fluorescence spectrophotometer. We preincubated HUVEC with or without nocodazole for 1h, and incubated for another 24h with ox-LDL, use Indirect Immunofluorescence to determine the location of LOX-1. We Knocked down cavoelin-1 by caveolin-1 siRNA in HUVEC, then determined the activation of NF-κB and expression of LOX-1.Result: We incubated HUVEC with ox-LDL(40μg/ml) for different times and depleted ox-LDL from medium after lipid-loaded 24h for different times, we found that HUVEC intra-cellular cholesterol contents increased in a time-dependented manner, and decreased gradually after depletion of ox-LDL from medium. We first incubated HUVEC with carrageenan, filipin, or nocodazole for 1h, and a second 24h incubation was performed after addition of DiI-ox-LDL(40μg/ml), we found that carrageenan, filipin, and nocodazole could inhibit ox-LDL uptake by HUVEC significantly. We also found that, after pretreated with those drugs, the Fluorescence intensity of DiI-ox-LDL that transcytosised up to the bottom reservoir was decreased dramatically to 49%, 72% and 80% respectively, comparing to control group. When used Immunofluorescence to locate LOX-1,we found that LOX-1 focus in endochylema, but when preincubated with nocodazole, then incubated with ox-LDL, LOCX-1 still focus at plasma membrane. After silenced the cavoelin-1 in HUVEC, we found that NF-κB was inactivated and the expression of LOX-1 was decreased.Conclusion1. HUVEC could uptake lipid, also could release lipid by some mechanism inherent exist, and this process is spontaneous. LOX-1 associated with caveolae in HUVEC, it located with or within cavoelae. Caveolae could be a carrier for ox-LDL involved in uptake of ox-LDL by HUVEC. HUVEC was a post-house in ox-LDL translocation from blood plasma to subendothelium, and cavoelae played a important role in this process.2. Ox-LDL up-regulated caveolin-1 expression, which triggered NF-κB activation and nuclear translocation, subsequent promoted LOX-1 gene transcription, at the end, LOX-1 expression up-regulated. In this pathway, caveolin-1 as a regulator play a important role in ox-LDL uptake by modulation of LOX-1’s expression in HUVEC.更多还原