医学毕业i型内皮素受体反义寡聚核苷酸抑制vsmc增殖及内膜增生.docxVIP

XX大学 毕业论文 I型内皮素受体反义寡聚核昔酸抑制VSMC增殖及内膜增生 姓 名： 2014年6月25日 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;反义寡核昔酸;血管平滑肌细胞 摘 要:目的 观察I型内皮素受体(ETA)反义寡核昔酸(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a-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，再对亍端硫代修饰 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;④运用放射受体结合分析法 (125I-ET-1) /解ODN对ETA蛋片表达的抑制作用，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 (3.0pmol L-l )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;15pmol 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以浓度和吋间依赖方式抑制ETA蛋 白表达以及VSMC的增殖;反义寡核昔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-| 1 receptor oligonucleotide in-| hibit vascular smooth muscle cell proliferation and intimal hyperplasia Abstract:AIM To evaluate the inhibitive effect of antisense cndothclin-1 receptor oligonucleotide ( ODN ) on VSMC growth and intimal hypcrplasia.METHODS ①Cell cul-turc.Thc medium of rabbit aorta were harvested, digested and the dispersions were cultured.The cells of passage4^6were routinely used to study after identification witha-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. (§) MTT method was used to evaluate DNA synthesis in ODN treated VSMC.④ 125 I-ET-1 radiobinding 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-l F-127gel solution at concentration of3.0|imol L-l 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