Purification of cartilage-derived anti-tumor factor (CATF) and establishment of cell lines which produce CATF

1.软骨衍生抗肿瘤因子（CATF）的纯化用1 M盐酸胍从胎牛软骨中提取CATF，并通过丙酮分级（45 - 65%）、超滤（分子量：100- 300 k）和DEAE-Sepharose CL-6 B柱层析（用0.3 - 0.35M NaCl在pH 8.0洗脱）进行部分纯化。部分纯化的CATF强烈抑制实体肉瘤180和B16黑色素瘤在体内的生长，牛肺动脉内皮细胞（CPAE）在培养中的增殖和DNA合成和B16黑色素瘤诱导的鸡胚绒毛尿囊膜（CAM）中的血管生成。用体外培养的CPAE细胞进行DNA合成抑制试验，结果表明，部分纯化的CATF的比活性约为粗品CATE的70倍.兔肋软骨细胞在原代培养中产生CATF通过暴露于原代培养的兔肋软骨细胞的无血清条件培养基特异性地抑制培养中的CPAE细胞的增殖和DBA合成。条件培养基（CM）中的因子也抑制B16黑色素瘤诱导的CAM中的血管生成和移植到CAM上的肿瘤的生长。这些结果强烈地表明，兔肋软骨细胞产生CATF。产CATF细胞系的建立从小鼠肋生长软骨的二次培养物中建立了一种显示生长软骨的一些表型的细胞系，如对甲状旁腺激素的反应性和高碱性磷酸酶活性。此外，从人软骨肉瘤中建立了显示典型软骨表型的细胞系。从这些细胞中获得的CM抑制CPAE细胞在培养中的增殖和DNA合成。对CPAE细胞有特异性抑制作用。因此，这些细胞系被认为产生CATF。

1. Purification of cartilage-derived anti-tumor factor (CATF)CATF was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and parrially purified by acetone fractionation (45 - 65%), ultrafiltration (Molecular weight; 100-300k) and DEAE-Sepharose CL-6B column chromatography (eluted with 0.3 to 0.35 M NaCl at pH 8.0). The partially purified CATF strongly inhibited the growth of solid sarcoma 180 and B16 melanoma in vivo, the proliferation and DNA synthesis of bovine pulmonary artery endothelial (CPAE) cells in culture and B16 melanoma-induced angiogenesis in chick embryo chorioallantoic membranes (CAM). The specific activity of this partially purified CATF was about 70 times that of crude CATE when determined using its inhibitory action on DNA synthesis in CPAE cells in culture.2. Production of CATF by rabbit costal chondrocytes in primary culture.Serum-free medium conditioned by exposure to rabbit costal chondrocytes in primary culture specifically inhibited the proliferation and DBA synthesis of CPAE cells in Culture. The factor in conditioned medium (CM) also inhibited angiohenesis in CAM induced by B16 melanoma and growth of the tumor transplanted onto the CAM. These findings strongly suggest that rabbit costal chondrocytes produce CATF.3. Establishment of cell lines producing CATF.A cell line showing some phenotypes of growth cartilage, such as responsiveness to parathyroid hormone and high alkaline phosphatase activity was established from secondary cultures of mouse costal growth cartilage. Moreover, a cell line showing typical cartilage phenotypes was established from human chondrosarcoma. CM obtained from these cells inhibited the proliferation and DNA synthesis of CPAE cells in culture. The inhibition was specific for CPAE cells. Therefore, these cell lines are thought to produce CATF.