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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。
OBJECTIVE: To search for small peptides that bind with high affinity and inhibit the biological function of ouabain, and provide a new strategy for the treatment of essential hypertension. Methods: Screening molecules with ouabain were screened with random 7-peptide library using M13 phage. After 3 rounds of bio-panning and identification by ELISA, phage-positive clones were identified by ssDNA electrophoresis and gene sequencing analysis. The binding activity of ouabain was evaluated by radioimmunoassay and radioimmunoassay. The inhibition of proliferation of vascular endothelial cells was detected by MTT assay. The morphological changes of cells were detected by Hoechst33342 / PI double staining. The expressions of α1 and β1 in sodium pump were detected by immunofluorescence. The changes of intracellular free Na + concentration were detected by fluorescent probe. Results: Fourteen positive clones were obtained by screening. Sequencing showed that the coincidence rate of ouabain binding peptides was 64.3% (9/14). Synthetic peptide (Arg-Cys-Met-Thr-Ser-Arg-Ser) was entrusted. Radioligand receptor binding assays show that the synthetic ouabain binding peptide binds to ouabain. The inhibitory rate of ouabain binding peptide + ouabain in EAhy926 group was less than that of ouabain group (P <0.05). Hoechst33342 / PI double staining showed that the cell death number was reduced. The expression of sodium pump α1 was detected by RT-PCR and immunocytochemistry , β1 subunit transcription and translation level, ouabain binding peptides can antagonize ouabain caused by the regulation of sodium pump α1 subunit expression, β1 subunit down-regulation; fluorescent probe shows that ouabain binding peptide can resist wow Inhibition of sodium pump by baine. CONCLUSION: ouabain-specific binding peptides can inhibit the inhibitory effect of ouabain on the proliferation and induction of death of vascular endothelial cells. In order to study the interaction mechanism between ouabain and sodium pump and to further study the molecular drugs of ouabain basis.