Micromethod of human thyrocyte cultures for detection of thyroid-stimulating antibodies and thyrotrophin.
Micromethod of human thyrocyte cultures for detection of thyroid-stimulating antibodies and thyrotrophin.
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用于检测促甲状腺抗体和促甲状腺激素的人甲状腺细胞培养物的微法。
DOI：
10.1530/acta.0.0970369
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
F. Seif
中科院分区：
文献类型：
--
作者：
G. Stöckle;R. Wahl;F. Seif
用原代细胞培养中人甲状腺细胞腺苷酸环化酶的激活和cAMP的释放检测Graves病患者血清中促甲状腺素（TSH）和促甲状腺抗体（TSAb）。组织消化和细胞分散使用多粘芽孢杆菌的中性蛋白酶（Dipase II）进行，它比胰蛋白酶收获更多重要的甲状腺细胞。这些细胞对刺激的反应增强了。用微试板代替培养瓶，提高了细胞制备和培养的效率。通过这种最小化，只需要0.6-1.8 X 10(6)个细胞来评估单个样品。环AMP浓度直接在上清培养基中通过竞争性蛋白质结合试验与木炭分离来测量。bTSH的最小检测剂量约为10微u /ml。随着bTSH剂量的增加，cAMP浓度上升至50 ~ 100 mU/ml左右的峰值，超过该峰值后cAMP逐渐下降，表明cAMP在激活机制中具有负协同作用。TSAb的“长效甲状腺刺激剂”作用表现为cAMP持续升高至最大值。41例甲状腺功能亢进合并Graves病患者血清tsab均为阳性，而中毒性甲状腺结节患者和甲状腺功能正常对照组血清tsab均为阴性。
The activation of adenylate cyclase of human thyrocytes in primary cell cultures and the release of cAMP into the medium are used to detect thyrotropin (TSH) and thyroid-stimulating antibodies (TSAb) in sera of patients with Graves' disease. Tissue digestion and cell dispersion are performed using a neutral protease of Bacillus polymyxa (Dipase II), which harvests more vital thyrocytes than does trypsin. The cells show an enhanced response to stimulation. The efficiency of the cell preparation and cultivation is increased by using microtest plates instead of culture flasks. By this minimization only 0.6-1.8 X 10(6) cells are needed to evaluate a single sample. Cyclic AMP concentrations are measured directly in the supernatant culture medium by a competitive protein binding assay with charcoal separation. The minimal detectable dose of bTSH is about 10 microU/ml. With increasing doses of bTSH cAMP concentrations rise to a peak at about 50 to 100 mU/ml, beyond which there is a gradual decrease of cAMP indicating negative cooperativity in the activating mechanisms. The 'long-acting thyroid stimulator' effect of TSAb is reflected by a protracted increase of cAMP to its maximal value. All 41 sera of hyperthyroid patients with Graves' disease were TSAb-positive, whereas sera of patients with toxic nodular goitre and euthyroid controls were TSAb-negative.