纤溶酶原Kringle5缺失突变体抑制血管内皮细胞增生的研究

A deletion mutant of plasminogen kringle 5 inhibits retinal capillary endothelial cell proliferation

目的纤溶酶原蛋白水解片段Kringle 5(K5)具有治疗血管增生性疾病的潜在临床应用价值,但是K5已获得美国专利。本研究应用基因突变和基因重组技术获得缺失突变型(K5Mut1)并检测其体外活性。方法除去K5外环两端的N端和C端15个氨基酸残基以降低其相对分子质量,但同时保留K5分子中的所有3个二硫键以保留完整的外环结构域,并在大肠杆菌中表达,亲和层析纯化,获得突变型K5 Mut1 (Cys1-80);MTT法分析突变型K5对牛视网膜毛细血管内皮细胞和周皮细胞的抑制作用。结果K5 Mut1以浓度依赖的方式抑制原代培养的视网膜内皮细胞,EC50(抑制50%细胞增生的药物浓度)约为35 nmol/L,是完整K5活性的2倍。在同样浓度范围内,K5 Mut1并不抑制同源的周皮细胞,表明K5 Mut1特异性抑制血管内皮细胞增生。结论K5 Mut1是一种比K5作用更强的血管增生抑制因子,在糖尿病性视网膜病、年龄相关性黄斑变性和实体肿瘤等血管增生性疾病的治疗中具有潜在的应用价值。

Objective To obtain purified deletion mutant of plasminogen kringle 5 (K5) using gene mutation and geneticrecombination methods and assess its anti-angiogenic activity in vitro. Methods A deletion mutant of K5 was obtained bydeleting 15 amino acids from K5 while retaining all the 3 disulfide bonds. This K5 mutant (Mut1) was expressed in E.coli and affinity purified. The inhibition effect of K5 Mut1 on primary retinal capillary endothelial cells and pericytes from thesame origin was assessed by MTT assay. Results The K5 Mut1 inhibited the proliferation of primary retinal capillary en-dothelial cells in a concentration-dependent manner, with an apparent half-inhibition concentration (EC50) of approximately 35 nmol/L, which was 2-fold more potent than intact K5. In the same concentration range, this peptide did not inhibit peri-cytes from the same origin, suggesting an endothelial cell-specific inhibition. Conclusion This K5 deletion mutant is amore potent angiogenic inhibitor than K5andmayhavetherapeuticpotentialinthetreatmentofsuchdisorderswithabnormalneovascularizationasdia-betic retinopathy, age-related macular degenerationand solid tumor.