Tarlow D M, Watkins P A, Reed R E, Miller R S, Zwergel E E, Lane M D
J Cell Biol. 1977 May;73(2):332-53. doi: 10.1083/jcb.73.2.332.
The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.
所述的非增殖性鸡肝细胞培养系统可产生具有供体动物生理营养状态特征的形态学和脂肪生成特性的细胞单层。脂肪酸、胆固醇和极低密度脂蛋白（VLDL）的合成与分泌以体内速率发生，并对影响体内这些过程的激素和试剂作出反应。如果培养基中存在胰岛素，来自喂食鸡的细胞可在数天内维持较高的脂肪酸和胆固醇合成速率。脂肪酸合成速率高与细胞质中出现膜包裹的富含甘油三酯的囊泡相关；去除胰岛素会导致脂肪酸合成活性降低（半衰期=22小时）。添加胰高血糖素或环磷酸腺苷（cAMP）会立即停止脂肪酸合成，并阻止富含甘油三酯的囊泡出现。由禁食鸡制备的肝细胞中的脂肪酸合成量不到喂食动物细胞的5%。在含有胰岛素+/-三碘甲状腺原氨酸的无血清培养基中培养2-3天后，脂肪酸合成恢复正常；胰高血糖素或二丁酰cAMP会阻止这种恢复。来自经雌二醇处理的鸡的肝细胞在培养中至少48小时合成并分泌VLDL。与未处理鸡的细胞相比，这些细胞的电子显微镜照片显示粗面内质网和高尔基体复合物有更广泛的发育。虽然[3H]亮氨酸掺入总蛋白不受雌激素处理的影响，但[3H]亮氨酸掺入细胞内和分泌的可免疫沉淀的VLDL中显著增加，表明VLDL载脂蛋白合成有特异性激活；合成并分泌的标记蛋白中有8-10%是VLDL。免疫沉淀的3H-VLDL的十二烷基硫酸钠-丙烯酰胺凝胶电泳显示出对应于纯化鸡VLDL的300,000、11,000和8,000道尔顿的三种主要载脂蛋白。
