Ikekubo K
Nihon Naibunpi Gakkai Zasshi. 1975 Oct 20;51(10):840-55. doi: 10.1507/endocrine1927.51.10_840.
The Leucocyte migration inhibiton test (LMT) using the agaraose plate technique according Clausen was performed in 19 patients with Hashimoto's thyroiditis, 23 patients with Graves' disease, and in 17 normal subjects. A series of leucocyte suspensions was preincubated at 37 degrees C for 30 min. with crude thyroid extract (Crude), purified thyroglobulin (Tg) and thyroid microsomes (Micr) in concentrations of 500, 300 and 100 muml, respectively together with phosphate buffer saline (PBS) as the control. Then, each of the aliquots of 7 mul of leucocyte suspension 1.5 million leucocytes, preincubated with various antigens of PBS, was placed into 4 wells, 2 in each of 2 agarose plates, followed by a 2nd incubation for 24 hours at 37 degrees C, in 2 per cent CO2 with 98 per cent atomospheric air, resulting in a pH of between 7.2 and 7.4. After the incubation, the migration areas were studied and measured by planimetry. The degree of migration inhibition was expressed as a migration index (MI); that is, the ratio of the average extent of migration of the leucocytes cultured with antigens to that of control. The MI values for normal subjects against Crude, Tg and Micr were 97.6 +/- 6.8 (mean +/- S.D)%, 97.8 +/- 4.2%, and 95.5 +/- 5.3%, respectively. Low MI values, below 2 S.D. of the normal, were regarded as positive results. The rate of positive LMT in patients with Hashimoto's thyroiditis were 32%, 46% and 31% against Crude, Tg and Micr, respectively. In Graves' disease, they were 35%, 50% and 41% for the same antigens, respectively. In 68--68% of patients with Hashimoto's thyroiditis or Graves' disease, positive LMT was at least shown with one of the three antigens. In Graves' disease, there was no correlation between MI values and the presence of long acting thyroid stimulator (LATS). In untreated patients with Hashimoto's thyroiditis and Graves' disease with a short clinical course, the migration inhibiton factor against Tg or Micr was found to be positive in a remarkably high rate. On the other hand, the levels of circulating antibodies in the serum tended to show high titers in patients with long duration, comparing with those with short duration. From the histological findings obtained by needle biopsy of thyroid tissue, grade of lymphocytic infiltration was found to be significantly correlated with the degree of reduction in MI values. From these observations, it is concluded that MIF against Tg can easily be detected by the agarose plate technique and that cellular immunity may play a more important role in the initial phase of autoimmune thyroid diseases than the later phase, in which the serum levels of circulating antibodies are becoming predominant.
采用克劳森的琼脂糖平板技术对19例桥本甲状腺炎患者、23例格雷夫斯病患者和17名正常受试者进行白细胞迁移抑制试验(LMT)。将一系列白细胞悬液在37℃下与浓度分别为500、300和100μg/ml的粗甲状腺提取物(粗提物)、纯化甲状腺球蛋白(Tg)和甲状腺微粒体(微粒体)预孵育30分钟,同时以磷酸盐缓冲盐水(PBS)作为对照。然后,将7μl白细胞悬液(150万个白细胞)的每个等分试样,与PBS或各种抗原预孵育后,分别放入4个孔中,每个琼脂糖平板放2个孔,接着在37℃、2%二氧化碳和98%大气空气条件下再孵育24小时,使pH值保持在7.2至7.4之间。孵育后,通过平面测量法研究并测量迁移区域。迁移抑制程度用迁移指数(MI)表示,即与抗原一起培养的白细胞平均迁移程度与对照的比值。正常受试者针对粗提物、Tg和微粒体的MI值分别为97.6±6.8(平均值±标准差)%、97.8±4.2%和95.5±5.3%。MI值低于正常均值2个标准差被视为阳性结果。桥本甲状腺炎患者针对粗提物、Tg和微粒体的LMT阳性率分别为32%、46%和31%。格雷夫斯病患者针对相同抗原的阳性率分别为35%、50%和41%。68%至68%的桥本甲状腺炎或格雷夫斯病患者至少对三种抗原中的一种呈现LMT阳性。在格雷夫斯病中,MI值与长效甲状腺刺激素(LATS)的存在之间无相关性。在未经治疗且病程较短的桥本甲状腺炎和格雷夫斯病患者中,针对Tg或微粒体的迁移抑制因子阳性率显著较高。另一方面,与病程短的患者相比,病程长的患者血清中循环抗体水平往往呈高滴度。从甲状腺组织针吸活检的组织学结果来看,淋巴细胞浸润程度与MI值降低程度显著相关。从这些观察结果得出结论,采用琼脂糖平板技术可轻松检测到针对Tg的迁移抑制因子,并且在自身免疫性甲状腺疾病的初始阶段,细胞免疫可能比后期发挥更重要的作用,后期血清中循环抗体水平占主导地位。