脂肪分化相关蛋白反义寡核苷酸降低乙酰辅酶A:胆固醇酰基转移酶活性

目的：已经发现随着细胞内胆固醇酯的积聚，脂肪分化相关蛋白的表达明显增加。本文进一步探讨脂肪分化相关蛋白干预细胞内胆固醇代谢的作用位点。 方法： Ox-LDL组为80 mg/L 氧化低密度脂蛋白与C57BL/6J小鼠腹腔巨噬细胞共同孵育，Ox-LDL+antisense组另加1 mmol/L脂肪分化相关蛋白反义寡核苷酸，在不同的时点取样，共观察4 d。使用荧光分光光度计观察细胞内胆固醇酯的改变；使用脂肪分化相关蛋白抗体间标法结合流式细胞术，观察脂肪分化相关蛋白表达量的变化；使用Ox-r［CL-3H］LDL和液体闪烁计数器观察细胞对氧化低密度脂蛋白摄入量的变化，并同时测定乙酰辅酶A:胆固醇酰基转移酶的活性。 结果： 72 h后，Ox-LDL+antisense组细胞内胆固醇酯（19.9±1.9）mg/g protein显著低于Ox-LDL组（46.6±3.4）mg/g protein。4 d观察期间，脂肪分化相关蛋白蛋白的表达量两组细胞都增加，从12 h时点开始，Ox-LDL组大于Ox-LDL+antisense组，在4 d时点，Ox-LDL组明显高于Ox-LDL+antisense组。两组细胞摄入Ox-r［CL-3H］LDL的量逐渐增加，但是，Ox-LDL+antisense组摄入量从1 d后明显低于Ox-LDL组，在2 d和4 d时点，两组差别仍有显著性。乙酰辅酶A:胆固醇酰基转移酶相对活性从6 h到2 d表现为增加，在2 d时点，两组比较差别显著，Ox-LDL+antisense组的活性明显低于Ox-LDL组。但从2 d到4 d，乙酰辅酶A:胆固醇酰基转移酶相对活性稳定。ACAT的活性与脂肪分化相关蛋白蛋白的表达量相关分析表明，Ox-LDL组R2＝0.6176 （P＜0.05），Ox-LDL+antisense组R2＝0.8212（P＜0.05）。 结论： 抑制脂肪分化相关蛋白表达能引起细胞摄入外源性脂蛋白减少和乙酰辅酶A:胆固醇酰基转移酶的活性降低，提示脂肪分化相关蛋白与脂滴的代谢功能密切相关，且乙酰辅酶A:胆固醇酰基转移酶有可能是其潜在的位点。

Adipophilin antisense oligonucleotides decrease ACAT activity

AIM: Based on the finding of adipophilin expression with the increase in cellular cholesterol, the aim of the present study was to look for the active site of adipophilin in cellular cholesteryl metabolism. METHODS: Mouse peritoneal macrophages were incubated with 80 mg/L Ox-LDL (Ox-LDL group) or 80 mg/L Ox-LDL plus 1 mmol/L adipophilin antisense oligonucleotides (Ox-LDL+antisense group), respectively. At the various time points, the incubated cell samples were observed with adipophilin immunofluorescence staining, flow cytometric analysis and cellular cholesterol analysis. RESULTS: The Ox-LDL+antisense group cells contained significantly lower cholesteryl ester (19.9±1.9) mg/g (protein) than that of cells in Ox-LDL group (46.6±3.4) mg/g (protein) at 4 days. From 12 h, expression of adipophilin in Ox-LDL group increased more quickly than that of the cells in Ox-LDL+antisense group. At day 4, the level of adipophilin expression in Ox-LDL group was significantly higher than that in Ox-LDL+antisense group. During the observation, the amount of Ox-r［CL-3H］ LDL taking up increased gradually in both groups, however, from day 1 the taking up amount in Ox-LDL+antisense group was less than that in Ox-LDL group. There was a statistical difference between the two groups from day 2 to day 4. From 6 h to day 2, the relative ACAT activity increased in both groups. The relative ACAT activity kept unchanged from day 2 to day 4 in the two groups. At day 2, the relative ACAT activity in Ox-LDL+antisense group was significantly lower than that in Ox-LDL group. Correlative analysis between activity of ACAT and adipophilin expression showed than R2 were 0.6176 and 0.8212 (P<0.05) in Ox-LDL group and Ox-LDL+antisense group, respectively. CONCLUSION: At least partly, expression of adipophilin is related to cellular taking up of lipoproteins and to ACAT activity. Therefore, the results indicate that adipophilin has a role in metabolism of cellular lipid droplets and the effective point may be ACAT.