17400 鲨鱼软骨血管生成抑制因子的纯化及生物学活性研究

Studies on Purification and Bioactivity of 17400 Shark Cartilage Angiogenesis Inhibitor

采用盐酸胍抽提、膜超滤、丙酮分级沉淀、Sephadex-75柱层析和C_4反相高效液相色谱等分离步骤,从广东阳江鲨鱼软骨中纯化获得新的鲨鱼软骨血管生成抑制因子SCAI-c(Shark Cartilage Angiogenesis In-hibitor-c,SCAI-c);SDS-PAGE电泳银染显示为一条带,根据蛋白质的相对迁移率计算,分子量为17 400;它对鸡胚绒毛尿囊膜血管的形成具有显著抑制效应,并有明显的浓度依赖关系.SCAI-c与SCAI-a具有类似的生物学特征.

SCAI-c, another angiogenesis inhibitor from shark cartilage of Guangdong Yangjiang, was purified and the bioactivity of SCAI-c were investigated. The 1. 0 mol/L guanidine and 0. 02 mol/L MES(at 28 ℃) extracts of shark cartilage was centrifuged at 6 000 r/min for 30 min and the pellet was discarded, and the supernatant was recentrifuged at 12 000 r/min for 30 min, then ultrafiltrated and fractionally precipitated with 25%-90% acetone. The precipitate was applied to a Sephadex G-75 gel column and eluted after the predominand peak of A280nm absorbing material with 0. 05 mol/L Tris-HCl buffer (containing 0. 02 mol/L NaCl and 0. 02% NaN3). The material B were composed of three single peptides with molecular mass of 17 600, 15 000, 12 600, then were purified by using reverse-phase HPLC, the eluate of C was collected, dried and observed by chicken chorioallantoic membrane. The pure inhibitor was homogeneous as a single band on a silver-stained 12. 5% sodium dodecyl sulfate-polyacrylamide gel. SCAI-c has a molecular weight of 17 600, and could specifically inhibit angiogenesis of the chorioallantoic membrane of chick embryos, and the inhibitory effect of angiogenesis was SCAI-c concentration dependent, but the concentration of SCAI-c increased to 0. 5 μg/μL, chick embryos died about to 20%-30%. Inhibitory characteristic of SCAI-c was similar with that of SCAI-a. They are specific angiogenesis inhibitor, and may be a useful drug on tumor therapy in some day.