反义adipophilin寡核苷酸降低ACAT活性(英)

已经发现，随着细胞内胆固醇酯的积聚，adipophilin的表达明显增加.在此基础上，为了寻找adipophilin在细胞内胆固醇代谢的作用点.小鼠腹腔巨噬细胞与80 mg/L OxLDL或80 mg/L OxLDL加1 mmol/L adipophilin反义寡核苷酸共孵育，在不同的时间点取样，使用免疫荧光染色，流式细胞仪分析和细胞内胆固醇测定.结果显示，72 h后，反义寡核苷酸处理组细胞内胆固醇酯显著下降到（19.9±1.9）mg/g，油红O染色显示，该组细胞质内红色脂滴明显减少.96 h的观察期间内，两组细胞adipophilin蛋白的表达量都增加.从12 h开始，未用反义寡核苷酸处理组adipophilin蛋白的表达量开始大于反义寡核苷酸处理组.在96 h时间点，统计学处理，未用反义寡核苷酸处理组明显高于反义寡核苷酸处理组，差别有显著性.使用Ox-r［CL-³H］LDL观察两组细胞摄入OxLDL的量，实验发现，两组细胞摄入Ox-r［CL-³H］LDL的量逐渐增加.但是，反义寡核苷酸处理组摄入Ox-r［CL-³H］LDL的量从24 h后明显低于未用反义寡核苷酸处理组，在48 h和96 h时间点，统计学处理，两组差别有显著性.观察ACAT的活性发现，ACAT相对活性从6 h到48 h表现为增加，但从48 h到96 h，ACAT相对活性趋于稳定.在48 h时间点，两组比较差别有显著性，反义寡核苷酸处理组的活性明显低于未用反义寡核苷酸处理组.相关分析发现，ACAT的活性与adipophilin蛋白的表达量有一定的联系，但不是直线相关.结果表明，adipophilin表达的高低与细胞摄入外源性脂蛋白，与ACAT的活性有一定的联系.提示adipophilin与脂滴的代谢功能密切相关，且ACAT有可能是其潜在的位点.

Adipophilin Antisense Oligonucleotides Decreased ACAT Activity

Based on the finding of adipophilin expression with the cellular cholesteryl ester increasing, the aim of study was to investigate the active site of adipophilin in cellular cholesteryl metabolism. Mice peritoneal macrophages were incubated with 80 mg/L OxLDL or 80 mg/L OxLDL plus 1 mmol/L adipophilin antisense oligonucleotides respectively. At various time points, the incubated cell samples were observed with adipophilin immunofluoresence staining, flow cytometric analysis and cellular cholesterol analysis. The results shown, the antisense oligonucleotides treated cells contained significantly lower cholesteryl ester ( 19. 9 ± 1.9) mg/g for 72 h. It was also shown by Oil Red O staining that the experimental group cells had less lipid droplets. During 96 h, expression of adipophilin increased in both groups. At 12 h, expression of adipophilin in OxLDL incubated cells increased quicker than that of the cells in antisense treated cells. At 96 h, the level of adipophilin expression in untreated control was significantly higher than that of antisense treated cells. The amount of OxLDL taking up by the cells was also observed with Ox-r CL-3H] LDL. During the observation, the amount of Ox-r CL-3H] LDL taking up increased gradually in both groups, however, from 24 h the taking up amount in antisense treated cells was less than that of untreated control. There was a statistical difference between the two groups from 24 h to 96 h. The activity of ACAT in the two groups was observed as well. From 6 h to 48 h, the relative ACAT activity increased in both groups. The relative ACAT activity kept unchanged from 48 h to 96 h in the two groups. At 48 h, the relative ACAT activity in antisense group was significantly lower than that of control group. The activity of ACAT was related to adipophilin expression but was not a linear relationship. This meant, at least partly, expression of adipophilin was related to cellular taking up of lipoproteins and to ACAT activity. Therefore, it is indicated that adipophilin has a role in metabolism of cellular lipid droplets and adipophilin is associated with ACAT.