核酸适配体对纤溶酶原Kringle 5抗血管生成功能的影响

Effect of Nucleic Acid Aptamers on the Anti-angiogenic Function of Plasminogen Kringle 5

关键词:  核酸适配体  纤溶酶原Kringle 5  特异性结合  细胞凋亡  血管生成

Key words:  nucleic acid aptamer  plasminogen Kringle 5  specific binding  apoptosis  angiogenesis

摘要:

目的 研究核酸适配体K-a2ct与纤溶酶原Kringle 5(K5)的特异性结合对K5抑制血管内皮细胞增殖和迁移以及促进血管内皮细胞凋亡功能的影响。方法 利用原核系统对重组K5蛋白进行克隆和表达，并采用亲和层析法对表达的K5蛋白进行分离纯化；使用等温滴定量热法(isothermal titration calorimetry，ITC)和酶联寡核苷酸吸附试验(enzyme-linked oligonucleotide adsorption assay，ELONA)对K-a2ct与K5的亲和特异性进行验证；采用CCK-8、细胞划痕试验观察K-a2ct对K5抑制人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVEC)增殖和迁移功能的影响；采用激光共聚焦显微镜观察Hoechst 33342染色的HUVEC细胞凋亡形态，并采用Annexin V/PI双染流式细胞术检测K-a2ct对K5促进HUVEC细胞凋亡功能的影响。结果 重组蛋白K5在大肠杆菌中实现了高效表达，经亲和层析纯化的重组K5相对分子质量为12 kDa，浓度为0.32 mg·mL^-1；ITC、ELONA结果显示K-a2ct对K5的亲和性强，且具有良好的选择性，具备典型核酸适配体的亲和特异性；CCK-8、细胞划痕试验结果表明，K-a2ct能够抑制K5的抗血管内皮细胞增殖和迁移的作用，且抑制程度与K-a2ct呈剂量依赖关系；激光共聚焦和流式细胞术结果表明，K-a2ct对K5促HUVEC细胞凋亡的功能有一定抑制作用，且主要影响K5作用的HUVEC细胞晚期凋亡，对早期凋亡影响不大。结论 核酸适配体K-a2ct以高亲和力和特异性结合K5，并以剂量依赖性方式抑制K5的抗血管生成功能，在靶向调节K5在体内的浓度和功能以及促进血管新生方面具有很大潜力。

Abstract:

OBJECTIVE To investigate the specific binding of nucleic acid aptamers(k-α2ct) with plasminogen Kringle 5(K5) on the function of K5 in inhibiting proliferation and migration of vascular endothelial cells and promoting their apoptosis. METHODS The cloning and expression of recombinant K5 protein were performed by using a prokaryotic system, and the isolation and purification of the expressed K5 protein were performed by affinity chromatography. The affinity and specificity of K-a2ct and K5 were verified using isothermal titration calorimetry(ITC) and enzyme-linked oligonucleotide adsorption assay(ELONA). The effect of K-a2ct on the function of K5 in inhibiting the proliferation and migration of human umbilical vein endothelial cells(HUVEC) was investigated by CCK-8 and cell scratch assay. The apoptotic morphology of HUVEC cells stained with Hoechst 33342 was observed by laser confocal microscopy, and the effect of K-a2ct on the apoptosis- promoting function of K5 in HUVEC cells was also examined by Annexin V/PI double-stained flow cytometry. RESULTS Recombinant protein K5 was efficiently expressed in Escherichia coli and purified by affinity chromatography, identified as having a relative molecular weight of 12 kDa and a concentration of 0.32 mg·mL^-1. ITC and ELONA results demonstrated that K-a2ct had a strong affinity and good selectivity for K5, showing the affinity specificity of a typical nucleic acid aptamer. CCK-8 and cell scratching assays showed that K-a2ct could inhibit the anti-proliferation and anti-migration effects of K5 on HUVEC cells in a dose-dependent manner. The laser confocal and flow cytometry results showed that K-a2ct inhibited the apoptosis-promoting function of K5 on HUVEC cells, mainly affecting late apoptosis of HUVEC cells effected by K5 but having little effect on early apoptosis. CONCLUSION The nucleic acid aptamers K-a2ct binds to K5 with high affinity and specificity, and inhibits its anti angiogenic function in a dose-dependent manner. It has great potential in targeting the regulation of K5 concentration and function in vivo and promoting angiogenesis.