Murakami S, Summer C N, Iida-Klein A, Anderson D G, Sugawara M
Medical, Service, Wadsworth Veterans Administration Hospital, Los Angeles, California 90073.
Endocrinology. 1990 Mar;126(3):1692-8. doi: 10.1210/endo-126-3-1692.
We describe a method of culturing intact porcine thyroid follicles for physiological de novo thyroid hormone formation; the roles of cAMP and protein kinase-C in thyroid hormone formation were also studied. Thyroid follicles were obtained by digesting minced porcine thyroid tissue with 0.04% collagenase and cultured in Coon's Modified Ham's F-12 medium supplemented with 0.5% calf serum, 0.5 mU/ml TSH, other standard hormones, and 3 antibiotics (6H medium). On the fourth day of culture, 6000-8000 follicles/well were plated in 12-well culture dishes. On the sixth day, thyroid hormone formation was carried out by incubating thyroid follicles with 0.5 microM KI in the presence of 6H medium for 2 days in a 5% CO2-95% air incubator at 37 C. To examine the effects of cAMP and protein kinase-C on de novo thyroid hormone formation, follicles were incubated with KI in the presence of 1-2.5 mM (Bu)2cAMP, 10 microM forskolin, 2 microM prostaglandin E2 (PGE2), or 0.5-1 microM 12-O-tetradecanoylphorbol-13-acetate in TSH-free medium for 2 days. The amount of newly formed thyroid hormone was measured by RIA of T3 content in the Pronase digest of thyroid follicular cells. Thyroid follicles cultured in 6H medium had normal polarity of the membrane, determined by electron microscope, and thyroid cAMP was responsive to the alteration of TSH. In this culture system cAMP alone was sufficient to form thyroid hormone. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase-C stimulator, disrupted thyroid follicles and inhibited cAMP-mediated thyroid hormone formation. The integrity of follicular structure was also required for thyroid hormone formation in this culture system. This study introduces perhaps the most physiological culture system for de novo thyroid hormone formation. Our data provide direct evidence that thyroid hormone formation is linked to cAMP and that the protein kinase-C system acts as an inhibitor of thyroid hormone formation.
我们描述了一种培养完整猪甲状腺滤泡以实现生理性从头合成甲状腺激素的方法；同时还研究了环磷酸腺苷（cAMP）和蛋白激酶-C在甲状腺激素合成中的作用。通过用0.04%胶原酶消化切碎的猪甲状腺组织获得甲状腺滤泡，并在补充有0.5%小牛血清、0.5 mU/ml促甲状腺激素（TSH）、其他标准激素和3种抗生素的库恩改良哈姆F-12培养基（6H培养基）中培养。在培养的第4天，将6000 - 8000个滤泡/孔接种到12孔培养板中。在第6天，通过在5%二氧化碳 - 95%空气的培养箱中于37℃在6H培养基存在下用0.5微摩尔碘化钾（KI）孵育甲状腺滤泡2天来进行甲状腺激素的合成。为了研究cAMP和蛋白激酶-C对从头合成甲状腺激素的影响，在无TSH的培养基中，将滤泡与KI一起在1 - 2.5毫摩尔二丁酰环磷腺苷（(Bu)2cAMP）、10微摩尔福斯可林、2微摩尔前列腺素E2（PGE2）或0.5 - 1微摩尔12 - O - 十四酰佛波醇 - 13 - 乙酸酯存在下孵育2天。通过放射免疫分析法（RIA）测定甲状腺滤泡细胞链霉蛋白酶消化物中三碘甲状腺原氨酸（T3）含量来测量新合成的甲状腺激素量。通过电子显微镜确定，在6H培养基中培养的甲状腺滤泡具有正常的膜极性，并且甲状腺cAMP对TSH的变化有反应。在这个培养系统中，单独的cAMP就足以合成甲状腺激素。蛋白激酶-C刺激剂12 - O - 十四酰佛波醇 - 13 - 乙酸酯破坏甲状腺滤泡并抑制cAMP介导的甲状腺激素合成。在这个培养系统中，甲状腺激素合成也需要滤泡结构的完整性。本研究介绍了一种可能是最生理性的从头合成甲状腺激素的培养系统。我们的数据提供了直接证据，表明甲状腺激素合成与cAMP有关，并且蛋白激酶-C系统作为甲状腺激素合成的抑制剂起作用。
