Stein Y, Stein O, Olivecrona T, Halperin G
Biochim Biophys Acta. 1985 May 17;834(3):336-45. doi: 10.1016/0005-2760(85)90007-4.
A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.
本文描述了一个用于研究胆固醇酯转运蛋白在间质胆固醇酯流出过程中假定作用的模型系统。通过与牛乳脂蛋白脂肪酶孵育，用[3H]胆固醇亚油酸醚和[14C]胆固醇亚油酸酯对牛主动脉平滑肌细胞的汇合培养物进行24小时标记。这种标记方法导致胆固醇亚油酸醚和胆固醇酯转移到三个部分：一个可被胰蛋白酶释放、对胰蛋白酶有抗性且可分解代谢的部分（斯坦因，O.，哈尔珀林，G.，莱特斯多夫，E.，奥利维克隆纳，T.和斯坦因，Y.（1984年）《生物化学与生物物理学学报》795，47 - 59）。在孵育后24小时内，研究了标记的胆固醇亚油酸醚和胆固醇酯从细胞外和细胞表面相关部分向含有1%牛血清白蛋白的无血清培养基中的流出情况。流出量以脉冲值的百分比表示，即标记期结束时细胞培养物中保留的放射性。[3H]胆固醇亚油酸醚、[14C]胆固醇酯和14C标记的游离胆固醇（由胆固醇酯的细胞水解形成）向含有1%牛血清白蛋白的培养基中的流出量约为脉冲值的5%。添加人缺乏脂蛋白的血清导致[3H]胆固醇亚油酸醚和[14C]胆固醇酯的流出量增加3 - 10倍，但对14C标记的游离胆固醇的流出量没有明显影响。不含胆固醇酯转运蛋白的大鼠缺乏脂蛋白的血清不会增加[3H]胆固醇亚油酸醚或[14C]胆固醇酯的流出量。在人缺乏脂蛋白的血清存在下，胆固醇酯的流出速率在约6小时内呈线性，并且在24小时内进一步增加。向含有人类缺乏脂蛋白血清的培养基中添加英脱利匹特进一步增强了[3H]胆固醇亚油酸醚的流出，对胆固醇酯的流出增强程度较小。向含有人类缺乏脂蛋白血清的培养基中添加大鼠极低密度脂蛋白也观察到了类似的效果。当向含有人类缺乏脂蛋白血清的培养基中添加大鼠高密度脂蛋白时，胆固醇亚油酸醚和胆固醇酯的流出受到抑制，游离胆固醇的流出显著增强，而人类高密度脂蛋白的抑制作用较弱。用人缺乏脂蛋白血清获得的结果用部分纯化的胆固醇酯转运蛋白得以重现。使用部分纯化的胆固醇酯转运蛋白，比较了胆固醇亚油酸酯和胆固醇油酸酯的流出情况，发现两者相同。
