摘要
目的:寻找与哇巴因高亲和力结合并抑制其生物功能的相关小分子肽,为治疗原发性高血压提供新策略。方法:以哇巴因为筛选分子,应用M13噬菌体呈现随机7肽库进行筛选。经过3轮生物淘筛,并通过ELISA法鉴定,对噬菌体阳性克隆ssDNA电泳鉴定和基因测序分析。委托合成筛选获得哇巴因结合肽,采用放射配基受体结合法检测其结合活性;MTT法检测血管内皮细胞的增殖抑制率,Hoechst33342/PI双荧光染色检测细胞形态学变化;利用RT-PCR和细胞免疫荧光检测钠泵α1、β1的表达水平;荧光探针检测细胞内游离Na+浓度变化。结果:筛选获得14个阳性克隆,基因测序显示:哇巴因结合肽一致率达64.3%(9/14)。委托合成肽(Arg-Cys-Met-Thr-Ser-Arg-Ser)。放射性配基受体结合法检测显示,合成的哇巴因结合肽能够与哇巴因结合。哇巴因结合肽+哇巴因的EAhy926细胞组增殖抑制率小于哇巴因组(P<0.05);Hoechst33342/PI双荧光染色显示细胞死亡数量减少;RT-PCR和免疫细胞化学检测钠泵α1、β1亚单位转录和翻译水平,显示哇巴因结合肽能拮抗哇巴因所引起钠泵α1亚单位表达的上调、β1亚单位的下调作用;荧光探针显示哇巴因结合肽能够拮抗哇巴因对钠泵的抑制作用。结论:哇巴因特异性结合肽能够阻抑哇巴因对血管内皮细胞的抑制增殖和诱导死亡作用,为研究哇巴因与钠泵的相互作用机制及进一步研究抗哇巴因的分子药物奠定基础。
AIM： To find one kind of peptide that will conjugate with ouabain and inhibit its biological function, and provide a new treatment strategy for primary hypertension. METHODS： In this study, ouabain was used as a target to screen ouabain conjugated peptide （OCP） from Ph. D.-7 phage display peptide library. After 3 rounds of bio-panning, the products were identified by ELISA and DNA electrophoresis analysis and sequencing. Peptide was synthesized and analyzed the activity by radioligand binding assay. The inhibitory ratio of cell proliferation was measured by MTT and the cell morphology changing was measured by Hoechst 33342/PI staining. The expression of Na^＋-K^＋-AT- Pase α1-subunit and β1-subunit were detected by RT-PCR and immunocytochemistry. The levels of the free intracellular Na^＋ in EAhy926 cells were measured by laser confocal microscope. RESULTS： The ouabain conjugated peptide was found out, and it was occupied in 0.64 （9/14）. The analysis of protein showed that the obtained peptides had no homology with Na^ ＋-K ^＋ -ATPase. The amino acid sequence of synthesized peptide was Arg-Cys-Met-Thr-Ser-Arg-Ser. There was binding activity between OCP and 3H.ouabain. The MTT assay showed that OCP could reverse the inhibition action of ouabain on vascular endothelial EAhy926 cells in a dose and time-dependent manner. The number of apoptotic cells had significantly decreased detected by Hoechst 33342/PI staining. The results of RT-PCR and immunocytochemistry showed that OCP could inhibit the up-regulated expression of Na ^＋ -K ^＋ -ATPase α1-subunit and down-regulated expression of Na^＋-K^＋-ATPase β1-subunit induced by ouabain in EAhy926 cells. CONCLUSION： The OCP could reverse the growth inhibition and death induction of ouabain in EAhy926 cells, which would provide the basis for studying the interaction between ouabain and Na^＋-K^＋ -ATPase and explore novel anti-ouabain agents.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2009年第10期870-874,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30840010)
天津市应用基础与前沿技术计划项目(06YFJMJC10400)
关键词
哇巴因
噬菌体展示肽库
结合肽
血管内皮细胞
细胞死亡
钠泵亚单位
ouabain
phage display peptide library
conjugated peptide
vascular endothelial cell
cell death
Na ^＋ -K^＋ -ATPase subunit
