日本七鳃鳗富含半胱氨酸RGD蛋白Lj-RGD1抑制血小板聚集及抗血管新生功能

Lj-RGD1来源于日本七鳃鳗口腔腺,富含半胱氨酸并具有RGD(Arg-Gly-Asp)模体.为了验证Lj-RGD1是否具有抑制血小板聚集、抗血管新生等去整合素样典型功能，本文对七鳃鳗Lj-RGD1进行了克隆表达及活性测定.提取七鳃鳗口腔腺中总RNA进行RT-PCR扩增，获得Lj-RGD1的420 bp cDNA.对其进行克隆、表达及组氨酸亲和层析纯化后，获得分子量为169 kD的可溶性蛋白rLj-RGD1.活性测定结果显示，rLj-RGD1呈剂量依赖方式抑制ADP诱导的兔血小板聚集，IC50为123 μmol/L；血管新生抑制实验结果表明，rLj-RGD1能够诱导ECV304细胞凋亡，抑制ECV304细胞增殖和管状物形成，并呈剂量依赖性方式抑制鸡胚胎绒毛尿囊膜（chorioallantoic membrane，CAM）血管新生.由此可见，Lj-RGD1具有去整合素样生物活性，在血栓或血管异常新生类疾病治疗中具有应用前景.

The Inhibition on Platelet Aggregation and Angiogenesis of rLj-RGD1,a Novel Cysteine-rich Protein with RGD Motif from Lampetra japonica

Lj-RGD1 was a novel protein derived from the buccal gland of Lampetra japonica, characterized with 14 cysteines and a RGD (Arg-Gly-Asp) motif which was found in most of the snake disintegrins. Sequence analysis showed that Lj-RGD1 has no similarity to any of the other disintegrins. In the present study, Lj-RGD1 was cloned and expressed in E. coli to study whether Lj-RGD1 inhibits platelet aggregation and anti-angiogenesis, as other disintegrins do. The soluble rLj-RGD1 was purified by using His- affinity chromatography with the molecular weight of 169 kD. The results showed that rLj-RGD1 inhibited rabbit platelet aggregation in a dose-dependent manner with IC50 value at 123μmol/L. In addition, rLj-RGD1 repressed bFGF-induced angiogenesis in vivo and in vitro, respectively, using ECV304 cells and chick chorioallantoic membrane angiogenesis models. These results suggested that rLj-RGD1 was a novel protein with typical disintegrin-like functions, which would be used as an anti-thrombosis or anti-angiogenesis drug in the future.