医学I型内皮素受体反义寡聚核苷酸抑VSMC增殖及内膜增生.docVIP

I型内皮素受体反义寡聚核苷酸抑制VSMC增殖及内膜增生 作者：钱济先 ，张柏根 ，李桂云 ，张岚，邓廉夫，朱雅萍 【关键词】 内皮素 　　关键词: 内皮素;反义寡核苷酸;血管平滑肌细胞 　　摘 要:目的 观察Ⅰ型内皮素受体（ETA）反义寡核苷酸（ODN）对血管平滑肌细胞（VSMC）增殖和内膜增生的抑制作用. 方法 ①细胞培养:取家兔主动脉中膜消化后培养传代，经抗α-actin鉴定后使用第4～6代传代细胞;②人工合成ODN:经Genbank检索筛选，合成含18bp ETA-ODN片段，再对5’端硫代修饰以增加稳定性，同时行地高辛标记以检测反义片段的细胞膜穿透性;③MTT分光光度法检测ETA-ODN对VSMC增殖的抑制作用;④运用放射受体结合分析法（125 I-ET-1）了解ODN对ETA蛋白表达的抑制作用，以探讨其反义作用;⑤兔颈动脉空气干燥模型在体研究ODN对血管内膜增生的抑制作用:应用F-127凝胶载体携带ETA-ODN（3.0μmol L-1 ）导入血管周围，术后不同时间观察增生内膜相对厚度. 结果 ODN于培养后12h进入细胞内，并逐渐增加;15μmol L-1 ETA-ODN对VSMC增殖具有抑制作用，24h后即有显著作用;ETA-ODN对ETA表达亦有明显的抑制作用，表现为CPM于24h逐渐减少;ETA-ODN导入后，血管增生内膜相对厚度减少. 结论 ETA-ODN以浓度和时间依赖方式抑制ETA蛋白表达以及VSMC的增殖;反义寡核苷酸是通过反义机制实现对ETA蛋白和VSMC增殖的抑制作用. 　　Antisense endothelin┐1receptor oligonucleotide in┐hibit vascular smooth muscle cell proliferation and intimal hyperplasia 　　Abstract:AIM To evaluate the inhibitive effect of antisense endothelin-1receptor oligonucleotide（ODN）on VSMC growth and intimal hyperplasia.METHODS ①Cell cul-ture.The medium of rabbit aorta were harvested，digested and the dispersions were cultured.The cells of passage4～6were routinely used to study after identification withα-actin antibody.②ODN synthesis.The antisense sequences were designed and screened through Genebank scanning.After these procedures，eighteen phosphorothioate antisense com-plementary to ETA cDNA were synthesized，and eventually labeled with digoxin by which the sequences stability be aug-mented and membranous permeability tested.③MTT method was used to evaluate DNA synthesis in ODN treated VSMC.④125 I-ET-1radiobinding assay to ETA and immuno-histochemistry were utilized to examine protein expression of the receptor，and to discuss roles of the ODN.⑤ODN dis-solved in200mL L-1 F-127gel solution at concentration of3.0μmol L-1 were applied to surround the exposed region of carotid artery on the basis of Air Dry Model.Thus the inti-mal thickness and luminal narrowness could be investigated.RESULTS ①ETA-ODN could be found in cultured VSMC in12h and gradually increased.②ETA-OD