I型内皮素受体反义寡聚核苷酸抑制VSMC增殖及内膜增生

I型内皮素受体反义寡聚核苷酸抑制VSMC增殖及内膜增生 I型内皮素受体反义寡聚核苷酸抑制VSMC增殖及内膜增生 I型内皮素受体反义寡聚核苷酸抑制VSMC增殖及内膜增生 I型内皮素受体反义寡聚核苷酸抑制VSMC增殖及内膜增生-医学论文 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?型内皮素受体反义寡核苷酸对血管平滑肌细胞增殖 和内膜增生的抑制作用. 方法 ?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;?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;?MTT分光光度法检测ETA-ODN对VSMC增殖的抑制作用; ?运用放射受体结合分析法了解ODN对ETA蛋白达的抑制作用，以 探讨其反义作用;?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 oligonucleotideon VSMC growth and intimal hyperplasia.METHODS ?Cell 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.?ODN 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.?MTT method was used to evaluate DNA synthesis in ODN treated VSMC.?125 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. ?ODN 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 ?ETA-ODN could be found in cultured VSMC in12h and gradually increased.?ETA-ODN at concentra-tion of15μmol L-1 suppressed VSMC proliferation，which showed a significant action in24h.?ETA protein expres-sion was reduced，indicated by the phenomenas that CPM progressively decreased in24h.?Intimal thickness and lu-minal narrowness were lightened after ETA-ODN induction.CONCLUSION ETA-ODN suppresses ETA expression and SMC proliferation in a time and concentration dependent manner.ETA plays an important role in mediating cultured VSMC proliferation course.ETA-ODN exerts inhibitory function through antisense mechanism.