Production of pancreatic secretory trypsin inhibitor (PSTI) as a growthstimulating factor and characterization of its specific receptors

作为生长刺激因子的胰腺分泌性胰蛋白酶抑制剂（PSTI）的生产及其特异性受体的表征

• 批准号: 63480135
• 负责人: OGAWA Michio
• 金额: $ 2.18万
• 依托单位: Osaka University Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480135/
• 关键词: pancreatic secretory trypsin inhibitor (PSTI); PSTI receptor; growth factor; PSTI受容体; 膵分泌性トリプシン・インヒビター(PSTI); EGFレセプター; 肺癌

Various cancer tissues contained pancreatic secretory trypsin inhibitor ( PSTI )-immunoreactive cells. Especially almost all adenocarcinorna tissues contained PSTI-immunoreactive cells, though the proportion of PSTI-positive cells varied in degree. Cultured human cells in protein-free nutrient medium secreted a considerable amount of PSTI into culture medium. The production of PSTI by cancer cells was confirmed by the isolation of PSTI cDNA clones from neoplastic tissues, the nucleotide sequence of neoplastic tissue PSTI cDNA being completely identical with that of pancreatic PSTI cDNA. Specific binding of ^<125>I-PSTI was noted with the following cells; WI-38, 3T3 Swiss albino, HUVE, BDC-1 and H4-lI-E-C3. Specific binding sites or human PSTI on 3T3 Swiss albino cells were studied using radioiodinated recombinant PSTI. On Scatchard analysis of the competitive binding data, linear plots indicated a single class of receptors with high affinity( Kd - 5.3 x 10^<-10>M ) on 3T3 Swiss albino … More cells, the number of receptors being 5,400 per cell. Treatment of surface bound radiolabeled PSTI with a chemical crosslinker ( disuccinimidyl suberate ) led to the identification of a membrane polypeptide of Mr 140,000 to which PSTI was crosslinked. The formation was inhibited by an excess amount of unlabeled PSTI in a dose dependent manner. The binding of ^<125>I-PSTI to 313 Swiss albino cells was competitively inhibited by unlabeled PSTI but not by other peptide hormones, such as EGF, b-FGF, IGF-I, TGF-alpha, PDGF and TNF, indicating the presence of receptors specific for PSTI. Various protease inhibitors had no or only a little effect, and mercaptoethanol and dithiothreitol strongly decreased the binding of ^<125>I-PSTI. Incubation at 37ﾟC resulted in rapid internalization of cell bound ^<125>I-PSTI, followed by the appearance of trichloroacetic acid-soluble ^<125>I-radioactivity in the culture medium, due to degradation of PSIT. In addition, PSTI stimulated [3^H]thymidine incorporation into DNA on 313 Swiss albino cells in a dose dependent manner. These results indicated that the biological effect of PSIT was mediated by high affinity plasma membrane receptors, which were not a cell-surface proteinase(s). Less

各种癌组织含有胰腺分泌性胰蛋白酶抑制剂（PSTI）免疫反应性细胞。尤其是几乎所有的腺癌组织都含有PSTI免疫反应性细胞，尽管PSTI阳性细胞的比例有不同程度的差异。在无蛋白营养培养基中培养的人体细胞会向培养基中分泌大量的 PSTI。通过从肿瘤组织中分离PSTI cDNA克隆来证实癌细胞产生PSTI，肿瘤组织PSTI cDNA的核苷酸序列与胰腺PSTI cDNA的核苷酸序列完全相同。注意到125 I-PSTI与以下细胞的特异性结合； WI-38、3T3 瑞士白化病、HUVE、BDC-1 和 H4-lI-E-C3。使用放射性碘标记的重组 PSTI 研究了 3T3 Swiss 白化细胞上的特定结合位点或人 PSTI。在对竞争性结合数据进行 Scatchard 分析时，线性图表明在 3T3 瑞士白化细胞上存在一类具有高亲和力 (Kd - 5.3 x 10^<-10>M) 的受体，每个细胞的受体数量为 5,400 个。用化学交联剂（二琥珀酰亚胺基辛二酸酯）处理表面结合的放射性标记的 PSTI，从而鉴定出与 PSTI 交联的 Mr 140,000 的膜多肽。过量的未标记 PSTI 以剂量依赖性方式抑制形成。 125 I-PSTI与313 Swiss白化细胞的结合被未标记的PSTI竞争性抑制，但不被其他肽激素例如EGF、b-FGF、IGF-I、TGF-α、PDGF和TNF抑制，表明存在PSTI特异性受体。各种蛋白酶抑制剂没有作用或仅有很小的作用，而巯基乙醇和二硫苏糖醇强烈降低125 I-PSTI的结合。 37℃孵育导致细胞结合的^<125>I-PSTI快速内化，随后由于PSIT的降解而在培养基中出现可溶于三氯乙酸的^<125>I-放射性。此外，PSTI 以剂量依赖性方式刺激 313 个瑞士白化细胞上的 [3^H] 胸苷掺入 DNA。这些结果表明 PSIT 的生物学效应是由高亲和力质膜受体介导的，而不是细胞表面蛋白酶。较少的

项目成果

小川道雄,他: "膵疾患と膵酵素-特に膵癌マ-カ-としての血中膵酵素について-" 臨床科学. 25. 606-615 (1989)

Michio Okawa 等人：“胰腺疾病和胰腺酶 - 特别是血液胰腺酶作为胰腺癌的标志物”《临床科学》25. 606-615 (1989)。

小川道雄: "「膵癌の診断と治療の進歩」血中膵酵素および酵素インヒビタ-による膵癌の診断-膵癌に患者における血中PSTI上昇とその意義-" 医学図書出版、土屋涼一他編, 19-21 (1989)

小川道夫：《胰腺癌诊断和治疗的进展》使用血液胰酶和酶抑制剂诊断胰腺癌 - 胰腺癌患者血液 PSTI 升高及其意义，医学东照出版社，土屋良一等编辑，19 -21 (1989)

Murata A., Ogawa M., Uda K., Matsuura N., Watanabe Y., Baba T., Mori T.: "Release of pancreatic secretory trypsin inhibitor from human hepatoblastoma cells on stimulation with cytokines." Life Sci., 43 : 1233-1240, 1988.

Murata A.、Okawa M.、Uda K.、Matsuura N.、Watanabe Y.、Baba T.、Mori T.：“细胞因子刺激后人肝母细胞瘤细胞释放胰腺分泌性胰蛋白酶抑制剂。”

Ueda G., Shimizu C., Tanaka Y., Inoue M., Tanizawa O., Ogawa M., Mori T.: "Immunohistochemical demonstration of pancreatic secretory trypsin inhibitor in gynecologic tumors." Gynecol. Oncol., 32 : 37-40, 1989.

Ueda G.、Shimizu C.、Tanaka Y.、Inoue M.、Tanizawa O.、Okawa M.、Mori T.：“妇科肿瘤中胰腺分泌性胰蛋白酶抑制剂的免疫组织化学论证。”

小川道雄: "異所性酵素産生腫瘍" BIOmedica. 5(3). 246-251 (1990)

小川道雄：“异位酶产生肿瘤”BIOmedica 5(3) (1990)。