Thrift R N, Forte T M, Cahoon B E, Shore V G
J Lipid Res. 1986 Mar;27(3):236-50.
Confluent monolayers of the human hepatoblastoma-derived cell line, Hep G2, were incubated in serum-free medium. Conditioned medium was ultracentrifugally separated into d less than 1.063 g/ml and d 1.063-1.20 g/ml fractions since very little VLDL was observed. The d less than 1.063 g/ml fraction was examined by electron microscopy; it contained particles of 24.5 +/- 2.3 nm diameter, similar in size to plasma LDL; a similar size was demonstrated by nondenaturing gradient gel electrophoresis. These particles possessed apoB-100 only. The d less than 1.063 g/ml fraction had a lipid composition unlike that of plasma LDL; unesterified cholesterol was elevated, there was relatively little cholesteryl ester, and triglyceride was the major core lipid. The d 1.063-1.20 g/ml fraction was heterogeneous in size and morphology. Electron microscopy revealed discoidal particles (14.9 +/- 3.2 nm long axis and 4.5 +/- 0.2 nm short axis) as well as small spherical ones (7.6 +/- 1.4 nm diameter). Nondenaturing gradient gel electrophoresis consistently showed the presence of peaks at 13.4 11.9, 9.7, and 7.4 nm. The latter peak was conspicuous and probably corresponded to the small spherical structures seen by electron microscopy. Unlike plasma HDL, Hep G2 d 1.063-1.20 g/ml lipoproteins contained little or no stainable material in the (HDL3a)gge region by gradient gel electrophoresis. Hep G2 d 1.063-1.20 g/ml lipoproteins differed significantly in composition from their plasma counterparts; unesterified cholesterol and phospholipid were elevated and the mole ratio of unesterified cholesterol to phospholipid was 0.8. Cholesteryl ester content was extremely low. ApoA-I was the major apolipoprotein, while apoE was the next most abundant protein; small quantities of apoA-II and apoCs were also present. Immunoblot analysis of the d 1.063-1.20 g/ml fraction after gradient gel electrophoresis showed that apoE was localized in the larger pore region of the gel (apparent diameter greater than 12.2 nm); the apoA-I distribution in this fraction was very broad (7.1-12.2 nm), and included a distinct band at 7.4 nm. Immunoblotting after gradient gel electrophoresis of concentrated medium revealed that a significant fraction of apoA-I in the uncentrifuged medium was in a lipid-poor or lipid-free form. This cell line may be a useful model for investigating the metabolism of newly formed HDL.
将人肝癌衍生细胞系Hep G2的汇合单层细胞在无血清培养基中培养。由于观察到的极低密度脂蛋白（VLDL）很少，条件培养基经超速离心分离成密度小于1.063 g/ml和密度为1.063 - 1.20 g/ml的组分。对密度小于1.063 g/ml的组分进行电子显微镜检查；它含有直径为24.5±2.3 nm的颗粒，大小与血浆低密度脂蛋白（LDL）相似；非变性梯度凝胶电泳也显示出类似大小。这些颗粒仅含有载脂蛋白B - 100。密度小于1.063 g/ml的组分的脂质组成与血浆LDL不同；游离胆固醇升高，胆固醇酯相对较少，甘油三酯是主要的核心脂质。密度为1.063 - 1.20 g/ml的组分在大小和形态上是异质的。电子显微镜显示出盘状颗粒（长轴14.9±3.2 nm，短轴4.5±0.2 nm）以及小的球形颗粒（直径7.6±1.4 nm）。非变性梯度凝胶电泳始终显示在13.4、11.9、9.7和7.4 nm处有峰。后一个峰很明显，可能对应于电子显微镜下看到的小的球形结构。与血浆高密度脂蛋白（HDL）不同，Hep G2密度为1.063 - 1.20 g/ml的脂蛋白在梯度凝胶电泳的（HDL3a）凝胶区域几乎没有或没有可染色物质。Hep G2密度为1.063 - 1.20 g/ml的脂蛋白在组成上与其血浆对应物有显著差异；游离胆固醇和磷脂升高，游离胆固醇与磷脂的摩尔比为0.8。胆固醇酯含量极低。载脂蛋白A - I是主要的载脂蛋白，而载脂蛋白E是其次最丰富的蛋白质；也存在少量的载脂蛋白A - II和载脂蛋白C。对梯度凝胶电泳后密度为1.063 - 1.20 g/ml的组分进行免疫印迹分析表明，载脂蛋白E位于凝胶的较大孔径区域（表观直径大于12.2 nm）；该组分中载脂蛋白A - I的分布非常宽（7.1 - 12.2 nm），并且在7.4 nm处有一条明显的条带。对浓缩培养基进行梯度凝胶电泳后的免疫印迹显示，未离心培养基中相当一部分载脂蛋白A - I处于脂质贫乏或无脂质形式。该细胞系可能是研究新形成的HDL代谢的有用模型。
