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Purification and Properties of a New Endotherium Growth Suppressing Factor from Macrophages

巨噬细胞中新型内皮生长抑制因子的纯化及其性质

基本信息

批准号：
11672176
负责人：
USUI Shigeyuki
金额：
$ 1.15万
依托单位：
Gifu Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2002
项目状态：
已结题

项目摘要

A carboxymethylated derivertives of a linear (1→3)-β-D-glucan (CMCD) acted directly on mouse peritoneal macrophages and mouse lymphoma P388D1 cells, and induced a growth suppressing activity for bovine artery endothelial cells (BAE) from themselves at a concentration of 100μg/mL. An endothelial cell growth-suppressing factor (EGSF) was purified from the conditioned medium of a mouse lymphoma P388D1 cell culture in the presence of 100μg/mL CMCD. The purification steps included, in order, ammonium sulfate fractionation and six stages of column chromatography. The purified EGSF showed two bands with silver staining on SDS-PAGE under reducing conditions and their molecular masses were estimated as approximately 55 and 63 kDa, while the molecular mass of the purified EGSF was estimated as about 65 kDa by gel filtration using Superdex 200HR. This factor strongly suppressed the proliferation of endothelial cells from bovine artery, human umbilical vein, and human dermal vas capillare and this … More suppression was observed to be reversible. The intravenous administration of the purified EGSF to sarcoma 180-bearing mouse caused a rapid decrease in the number of viable tumor cells in tumor lumps within 16h. Immunohistochemical study demonstrated that hemorrhagic disorder all over the tissue in the tumor lamp occurred under this circumstance. Thus, the purified factor exhibited not only growth suppressing activity for endothelial cells but also tumor regressing activity. The purified factor significantly inhibited in vitro tubulogenesis of bovine artery, human umbilical vein, and adult human darmal microvascular endothelial cells on collagen gel. After the tube formation of endothelial cells was completed on a collagen gel, the purified factor disrupted the tubes at a concentration of about 5 ng/ml within 48h. These findings demonstrate that EGSF is a potent inhibitor of angiogenesis as well as the growth of endothelial cells, and may bring about the regression of a solid tumor by inhibiting angiogenesis. Less

线性 (1→3)-β-D-葡聚糖 (CMCD) 的羧甲基化衍生物直接作用于小鼠腹腔巨噬细胞和小鼠淋巴瘤 P388D1 细胞，并在浓度为 100μg/mL 时诱导牛动脉内皮细胞 (BAE) 生长抑制活性。在 100μg/mL CMCD 存在下，从小鼠淋巴瘤 P388D1 细胞培养物的条件培养基中纯化出内皮细胞生长抑制因子 (EGSF)。纯化步骤依次包括硫酸铵分级分离和六级柱色谱。纯化的EGSF在还原条件下在SDS-PAGE上显示出两条银染条带，并且它们的分子量估计为大约55和63kDa，而通过使用Superdex 200HR的凝胶过滤估计纯化的EGSF的分子量为大约65kDa。该因子强烈抑制牛动脉、人脐静脉和人真皮毛细血管内皮细胞的增殖，并且观察到这种抑制是可逆的。将纯化的EGSF静脉注射给携带肉瘤180的小鼠，在16小时内导致肿瘤肿块中活肿瘤细胞的数量迅速减少。免疫组化研究表明，在这种情况下，肿瘤灯内出现了全身组织的出血性疾病。因此，纯化的因子不仅表现出内皮细胞生长抑制活性，而且表现出肿瘤消退活性。纯化的因子显着抑制胶原凝胶上牛动脉、人脐静脉和成人真皮微血管内皮细胞的体外管状形成。内皮细胞在胶原凝胶上成管完成后，纯化因子在48小时内以约5ng/ml的浓度破坏管。这些发现表明EGSF是血管生成以及内皮细胞生长的有效抑制剂，并且可能通过抑制血管生成来使实体瘤消退。较少的