A new in vitro assay for human thyroid stimulator using cultured thyroid cells: effect of sodium chloride on adenosine 3',5'-monophosphate increase.
A new in vitro assay for human thyroid stimulator using cultured thyroid cells: effect of sodium chloride on adenosine 3',5'-monophosphate increase.
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使用培养的甲状腺细胞进行人甲状腺刺激剂的新体外测定：氯化钠对腺苷 3,5-单磷酸增加的影响。
DOI：
10.1210/jcem-54-1-108
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
K. Torizuka
中科院分区：
文献类型：
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作者：
K. Kasagi;J. Konishi;Y. Iida;K. Ikekubo;T. Mori;K. Kuma;K. Torizuka
使用单层培养的人甲状腺腺瘤细胞开发了一种新的人甲状腺刺激剂（HTS）灵敏体外测定法。培养2天后，将细胞在0.3ml不含0.8％NaCl的Hank's溶液（培养基1）和甲状腺刺激剂（牛TSH或3mg患者血清免疫球蛋白G）中于37℃温育2小时。通过RIA测量培养期间细胞和培养基中产生的cAMP。该测定足够灵敏，在 TSH 浓度为 10 microU/ml 时，cAMP 增加 1.7 至 7.9 倍。使用该检测系统，在 40 名未经治疗的格雷夫斯病患者中，有 33 名 (82.5%) 检测到了 HTS。然而，在 Hank 溶液（介质 2）中，21 名未经治疗的 GRaves 病患者中仅 5 名（23.8%）检测到 HTS。培养基1中受TSH或HTS刺激后的cAMP增量大于培养基2中的cAMP增量，并且使用两种培养基对HTS的反应的差异远大于对TSH的反应的差异。因此，所有 HTS-免疫球蛋白 G 研究均表明，当以牛 TSH 当量表示时，使用培养基 1 的活性高于使用培养基 2 的活性。通过 Lineweaver-Burk 图对 TSH 和 HTS 刺激对 cAMP 增量的影响的剂量反应曲线进行分析，结果表明，在培养基中添加 NaCl 后，Km 增加。在使用粗质膜级分的人甲状腺腺苷酸环化酶刺激剂的测定系统中也观察到了 NaCl (150 mM) 的类似抑制作用。总之：1) 开发了一种测量培养的甲状腺腺瘤细胞中 cAMP 产生的 HTS 测定方法，并且发现使用低 NaCL 培养基的测定方法是最敏感的，2) NaCl 对 HTS 反应的抑制作用远大于对 TSH 反应的抑制作用。这些数据表明这两种刺激器在其受体位点的不同行为。
A new sensitive in vitro assay for human thyroid stimulator (HTS) was developed using human thyroid adenoma cells in monolayer culture. After being cultured for 2 days, the cells were incubated in 0.3 ml Hank's solution without 0.8% NaCl (medium 1) and with thyroid stimulator (bovine TSH or 3 mg patient serum immunoglobulin G) at 37 C for 2 h. The cAMP generated in the cells and the medium during the incubation was measured by RIA. The assay was sensitive enough to elicit a 1.7- to 7.9-fold increase in cAMP at a TSH concentration of 10 microU/ml. HTS was detected in 33 (82.5%) of the 40 patients with untreated graves' disease using this assay system. In Hank's solution (medium 2), however, HTS was detected in only 5 (23.8%) of the 21 patients with untreated GRaves' disease. cAMP increment upon stimulation by either TSH or HTS in medium 1 was larger than that in medium 2, and the difference in the response to HTS using the two media was much greater than that in the response to TSH. Therefore, all HTS-immunoglobulin G studies showed higher activity using medium 1 than using medium 2 when expressed as bovine TSH equivalent. Analysis by the Lineweaver-Burk plot of dose-response curves of the effect of TSH and HTS stimulation on cAMP increment showed an increase in the Km upon the addition of NaCl to the medium. A similar inhibitory effect of NaCl (150 mM) was also observed in the assay system of human thyroid adenylate cyclase stimulator using crude plasma membrane fractions. In summary: 1) an assay for HTS measuring cAMP production in cultured thyroid adenoma cells was developed and the assay using low NaCL medium was found to be the most sensitive, and 2) the inhibitory effect of NaCl on the response to HTS was much greater than that on the response to TSH. These data suggest different behaviors of these two stimulators at their receptor sites.