갑상선자극호르몬이갑상선암세포의 VEGF, 신생혈관형성, 성장, 침윤및전이에미치는영향 Effects of TSH on the Production of VEGF, Angiogenesis, Growth, Invasion and Metastasis in Thyroid Cancer Cells Euy Young Soh, M.D., Hee Boong Park, M.D., Hye Jin Kim, M.D. and Hyun Man Kim, M.D. 1 Purpose: Vascular endothelial growth factor (VEGF) is a vascular endothelial cell specific mitogen and a major regulator of angiogenesis. VEGF secretion is activated in some thyroid cancers and that VEGF secretion is stimulated by TSH. So we postulated that TSH may promote growth and invasion in some thyroid cancers by stimulating VEGF secretion and angiogenesis. Methods and Results: We investigated the TSH effect for the VEGF secretion, endothelial cell proliferation and invasion in vitro with the primary cultured normal thyroid cell (NT-1) and thyroid cancer cell line (TPC-1). And to evaluate the relationship between TSH and VEGF, angiogenesis and tumor growth in vivo, we xenografted human dermal matrix inoculated with thyroid cells into nude mice or directly injected subcutaneously. For the study, mice were made hypothyroid (Group 1) by antithyroid hormone p.o, hyperthyroid (Group 2) by L-thyroxine injection and euthyroid (Group 3). One week after the treatment, significant difference were noted in T3, T4 and TSH level between each group, but the VEGF level showed significant difference in group 1 only compared with group 2 and 3. NT-1 or TPC-1 were seeded in the upper chamber of Transwell and HUVEC were cultured in lower chamber, and added different concentration of TSH. NT-1 and TPC-1 secreted VEGF under basal condition, but the level were similar. TPC-1 cells secreted significantly more VEGF than NT-1 after TSH (1, 10, 100 miu/dl) stimulation, which were also parallel with the concentration of TSH. In low concentration of TSH (0, 1 miu/dl), there were no difference of HUVEC proliferation between NT-1 and TPC 1. In high concentration of TSH (10, 100 miu/dl), however, TPC-1 enhanced HUVEC proliferation than NT-1 significantly (p 0.05). Similar findings were noted in thyroid cell invasion. Invasion was higher in TPC-1 than in NT-1 in high concentration of TSH (10, 100 miu/dl). In vivo study using the dermal matrix showed that number of blood vessels ingrowth were higher in Group 1 (25/HPF) than Group 2 (16/HPF) or Group 3 (17/HPF). But there was no difference between Group 2 and Group 3. Level of TSH and VEGF were also increased significantly in Group 1 compared with in Group 2 and Group 3. The size of tumor did not showed significant difference between each group during observation. The tumor from Group 1 (6.2 gm) were larger compared with Group 2 (5.1 gm) or Group 3 (5.6 gm), but this difference was not significant statistically (p 0.05). The number of blood vessels in tumor were also more increased in Group 1 and were commonly located in the peripheral portion of tumor. Conclusion: We conclude that thyroid cancer cell line secrete the VEGF and TSH secretion is more enhanced by the stimulation of TSH. And increased VEGF promote the vascular endothelial cell proliferation, invasion and angiogenesis in thyroid cancer. (Korean J Endocrine Surg 2001; 1:51-60) Key Words: Thyroid stimulating hormone (TSH), Angiogenesis, Vascular endothelial growth factor (VEGF), Thyroid cancer, Dermal matrix : Departments of Surgery and 1 Endocrinology, Ajou University School of Medicine, Suwon, Korea
56 대한내분비외과학회지 제 1 권 제 1 호 2001 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ 과 3군 사이에는 차이가 없었다(Table 4). 4) Angiogenic effect of TSH in dermal matrix Mice에 이식한 dermal matrix에서 새로운 혈관의 형성은 전층에서 관찰되었다. H&E 염색상 적혈구가 혈관내에 있 는 것을 신생혈관으로 간주하였으며 임으로 가장 혈관의 밀도가 높은 곳을 5군데 선정하여 그 수를 측정하였다. 1 군의 평균 혈관의 수는 25.0±4.63개/HPF (mean±sd)로 2 군 16.2개/HPF과 3군 17.2개/HPF에 비하여 의의있게 신생 혈관의 수가 증가되어 있었다(p 0.05). 2군과 3군간에는 의의있는 차이를 보이지 않았다. 2주째 VEGF는 1군이 5.35±1.98 ng/dl로 2군 및 3군에 비하여 의의있는 차이를 보이고 있었다(Table 5)(Fig. 1). 5) Tumor growth effects of TSH in nude mice 실험 기간 동안 mice의 몸무게는 의의있는 차이는 보이 지 않았다. 각군에 이식한 종양은 기간이 지남에 따라 점 차 크기가 증가하였으나 세군간에 의의있는 크기의 차이 는 없었다. 이식 4주째 쥐를 사망시킨 후 종양의 무게를 측정하였으며 1군(6.24±87 gm)이 2군(5.1±0.91 gm) 및 3군 (5.58±0.93 gm)에 비하여 무게가 컸으나 세군간에 의의있 는 차이는 보이지 않았다(p 0.05)(Fig. 2). 그러나 VEGF 치 는 1군이 2군 및 3군에 비하여 의의있게 증가되어 있었다. 모든 실험쥐에서 복강내 장기의 전이 유무를 조사하였으나 전이는 없었다. 종양 조직을 H&E 염색 후 종양내의 혈관을 관찰하였으며 1군이 2군 및 3군에 비하여 종양의 표면에 신 생혈관의 수가 증가되어있음을 관찰할 수 있었다(Fig. 3). Table 5. Effects of thyroid function on angiogenesis and VEGF level in nude mice ꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚ Group 1 Group 2 Group 3 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Vessel density 25.0±4.63* 16.2±3.4 17.2±2.6 VEGF 5.35±1.98* 1.1±0.23 1.5±0.56 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Values are mean±sd. Vessel density: No. of blood vessel/hpf. VEGF: ng/dl. * = p 0.05 between Group 1 and Group 2 or Group 3. A B C Fig. 1. H&E staining of human dermal matrix in hypothyroid (A), hyperthyroid (B) and euthyrooid (C) rat. Arrows denote staining of new blood vessels with RBC ( 100).
소의영 외 갑상선 자극호르몬이 갑상선암세포의 VEGF, 신생혈관 형성, 성장, 침윤 및 전이에 미치는 영향 57 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ A B Fig. 2. Tumor mass in nude mice injected with TPC-1, A: 2 weeks after xenograft, B: 4 weeks after xenograft. Hypothyroid mice (upper), hyperthyroid mice (middle) and euthyrooid mice (lower). A B C Fig. 3. H&E staining of tumor in hypothyroid (A), hyperthyroid (B) and euthyrooid (C) rat. Arrows denote staining of new blood vessels with RBC ( 100). 고 찰 악성종양에서 갑상선암이 차지하는 비율은 1 2%에 불 과하나 내분비암 중에서는 가장 발생 빈도가 높다. 갑상 선암은 오랫동안 indolent 상태를 유지하거나 천천히 자라 서 좋은 경과를 보이기도하나 때로는 급격히 성장하고 주 위의 주요 장기에 침윤 및 원격전이를 일으키는 등 다양한