重组血管生成抑制因子r-K4K5的分离纯化与功能鉴定

摘要

Purpose To purify recombinant angiogenesis inhibitor r-K4K5 and investigate its inhibitoryeffects on bovine capillary endothelial (BCE) cell proliferation, chick embryo chorioallantoic membrane(CAM) angiogenesis and growth of experimental human non-small cell lung cancer (adeno). Methodsr-K4K5 was obtained by salting out and gel filtration with the purity of 95% determined by SDS-PAGE.BCE cells were cultured with DMEM media containing r-K4K5. The cells were counted in 24,48,72 hrespectively. r-K4K5 was injected daily into all 7-day chick embryo CAMs and CAM angiogenesis wasobserved at 72 h after incubation. The Balb/c (nuu) mice implanted with human SPC-Al tumor pieceswere grown for 10 days and then randomly divided into three groups. One group was treated with PBS, theother two groups were treated with local subcutaneous injection of purified r-K4K5 at 8 μg and 80 μg lpermouse every other day. They were daily observed and sacrificed in 14 days. Each tumor was weighed.Results The number of BCE cells, blood vessels diameter less than 50 μn of chick embryo CAM and theaverage weight of experimental tumor were decreased markedly in all the groups treated with r-K4K5.Conclusions r-K4K5 inhibits proliferation of BCE cells, angiogenesis of chick embryo CAMs and thegrowth of experimental human SPC-A1 non small lung cancer (adeno).%目的 分离纯化r-K4K5，探讨r-K4K5对牛毛细血管内皮(BCE)细胞、鸡胚绒毛尿囊膜(CAM)新生血管生成及实验性人肺腺癌SPC-Al生长的抑制作用。方法 通过盐析、凝胶过滤提纯r-K4K5，BCE细胞在含r-K4K5的DMEM中培养24、48、72 h后分别计数；孵化7 d的鸡胚加r-K4K5后继续孵育72 h，观察新生血管生成；已经接种人SPC-Al肺腺癌组织的荷瘤裸小鼠(Balb/c，nuu)，瘤旁注射r-K4K5继续饲养，观察肿瘤生长变化。结果 r-K4K5抑制BCE细胞增殖，48～72 h作用明显；r-K4K5处理的CAM组中直径小于50μm的小血管明显减少；高剂量r-K4K5治疗的荷瘤裸小鼠组，平均瘤重与对照组比较有统计学意义。结论 r-K4K5能够抑制BCE细胞增殖，抑制鸡胚CAM新生血管生成，抑制实验性人SPC-Al肺腺癌生长。