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

1.软骨源性抗肿瘤因子(CATF)的纯化用1M盐酸胍从胎牛软骨中提取，经丙酮分级(45-65%)、超滤(100-300K)和DEAE-SepharoseCL-6B柱层析(0.3-0.35M氯化钠在pH 8.0下洗脱)进行初步纯化。部分纯化的CATF对实体肉瘤180和B16黑色素瘤的体内生长、体外培养的牛肺动脉内皮细胞的增殖和DNA合成以及B16黑色素瘤诱导的鸡胚绒毛尿囊膜血管生成均有明显的抑制作用。结论：1.部分纯化的CATF对体外培养的CPAE细胞DNA合成有抑制作用，其比活力约为粗提物的70倍。兔肋软骨细胞原代培养产生CATF，经原代培养的兔肋软骨细胞作用的无血清培养基能特异性抑制CPAE细胞的增殖和DBA的合成。条件培养液中的因子(CM)也能抑制B16黑色素瘤诱导的CAM血管生成和移植到CAM上的肿瘤生长。这些发现强烈表明，兔肋软骨细胞产生CATF.3。从小鼠肋骨生长软骨传代培养中建立了对甲状旁腺激素敏感、碱性磷酸酶活力较高的生长软骨细胞系。此外，从人软骨肉瘤中建立了具有典型软骨表型的细胞系。从这些细胞中获得的CM可抑制体外培养的CPAE细胞的增殖和DNA合成。该抑制作用对CPAE细胞具有特异性。因此，这些细胞系被认为能产生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.