Tam S P, Ramharack R
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Atherosclerosis. 1992 Aug;95(2-3):137-46. doi: 10.1016/0021-9150(92)90017-b.
The human hepatoma cell line, HepG2, was cultured with 25 OH cholesterol, a potent inhibitor of HMG-CoA reductase, in order to examine the effect of the oxysterol on apo E synthesis and secretion. Treatment of cells with oxysterol (2.5 microM) resulted in a greater than 90% inhibition of HMG-CoA reductase activity and a 3-fold reduction in its cognate mRNA level. However, apo E mRNA level and secretion were not affected after 24 h of drug treatment. This drug treatment was associated with a reduction in both cellular free and esterified cholesterol levels by 50% and 40%, respectively. Exposure of HepG2 cells to an ACAT inhibitor, the Sandoz compound (58-035) for 24 h, at a concentration of 5 micrograms/ml, resulted in a 30% increase and 70% decrease in the intracellular levels of free and esterified cholesterol, respectively. Under this regimen of drug treatment, the level of apo E mRNA was increased by approximately 70%, while HMG-CoA reductase mRNA level was decreased by 35%. When the cells were exposed to the combination of the ACAT inhibitor and 25 OH cholesterol, the cellular levels of free and esterified cholesterol were reduced by 30% and 80%, respectively. This combination of drugs had no effect on apo E mRNA; however, the level of HMG-CoA reductase mRNA was decreased by 3.5-fold. Taken together, the data suggested that reduction in the intracellular levels of either free or esterified cholesterol had no effect on apo E mRNA level. By contrast, a small increment in cellular free cholesterol content was associated with a significant induction in apo E mRNA level. Furthermore, 25 OH cholesterol caused a significant redistribution (50%) of apo E from the HDL fraction to the d greater than 1.21 g/ml infranatant. By using high performance liquid chromatography and molecular sieve columns, it was found that the appearance of a lipid-poor apo E particle was not an artifact of ultracentrifugation. This particle contained 85 wt% protein and 15 wt% of free cholesterol and phospholipid. The results suggested that a lipid-poor apo E particle was secreted by the HepG2 cells under certain circumstances.
使用3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶的强效抑制剂25-羟基胆固醇培养人肝癌细胞系HepG2，以研究氧化甾醇对载脂蛋白E（apo E）合成与分泌的影响。用氧化甾醇（2.5微摩尔）处理细胞导致HMG-CoA还原酶活性受到超过90%的抑制，其相应的信使核糖核酸（mRNA）水平降低了3倍。然而，药物处理24小时后，apo E的mRNA水平和分泌未受影响。这种药物处理分别使细胞内游离胆固醇和酯化胆固醇水平降低了50%和40%。将HepG2细胞暴露于一种酰基辅酶A：胆固醇酰基转移酶（ACAT）抑制剂，即山德士化合物（58-035），浓度为5微克/毫升，处理24小时，导致细胞内游离胆固醇水平增加30%，酯化胆固醇水平降低70%。在这种药物处理方案下，apo E的mRNA水平增加了约70%，而HMG-CoA还原酶的mRNA水平降低了35%。当细胞暴露于ACAT抑制剂和25-羟基胆固醇的组合时，细胞内游离胆固醇和酯化胆固醇水平分别降低了30%和80%。这种药物组合对apo E的mRNA没有影响；然而，HMG-CoA还原酶的mRNA水平降低了3.5倍。综上所述，数据表明细胞内游离或酯化胆固醇水平的降低对apo E的mRNA水平没有影响。相比之下，细胞内游离胆固醇含量的小幅增加与apo E的mRNA水平的显著诱导相关。此外，25-羟基胆固醇导致apo E从高密度脂蛋白（HDL）组分向密度大于1.21克/毫升的下层组分发生显著重新分布（50%）。通过使用高效液相色谱法和分子筛柱，发现脂质含量低的apo E颗粒的出现不是超速离心的假象。该颗粒含有85%（重量）的蛋白质以及15%（重量）的游离胆固醇和磷脂。结果表明，在某些情况下，HepG2细胞会分泌脂质含量低的apo E颗粒。
