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  

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

作　　者：王继红[1,2] 于凤[1,2] 韩晓曦[1] 吕莉[3] 刘欣[1,2] 李庆伟[1,2] 

机构地区：[1]辽宁师范大学生命科学学院,辽宁大连116029 [2]辽宁省生物技术与分子药物研发重点实验室,辽宁大连116029 [3]大连医科大学药学院药理教研室,辽宁大连116044

出　　处：《中国生物化学与分子生物学报》2012年第6期531-537,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家高技术研究发展计划(863计划;No.2007AA09Z428);国家自然科学基金(No.30770297);辽宁省教育厅创新团队项目(No.2008T102);辽宁省科学技术计划项目(No.2010402009);大连市重大科技攻关项目(No.2009E11SF127;No.2010E13SF143)~~

摘　　要：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420bp 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12.3μmol/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.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. Inthe 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 ICso value at 12.31xmol/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 druz in the future.

关 键 词：日本七鳃鳗 Lj-RGD1 去整合素 血小板聚集 血管新生 

分 类 号：Q781[生物学—分子生物学]