【摘要】 目的 研究血管紧张素Ⅰ型受体(AT1)反义(AS)寡核苷酸(ODN)对大鼠肾脏局 部肾素-血管紧张素系统(RAS)的阻断作用。方法 采用单侧肾动脉注射+原位电穿孔的方法, 将AT1 AS-ODN导入到Wistar大鼠的一侧肾脏,观察以FITC荧光标记的ODN在肾脏的转移位 置,以AT1放射自显影定量分析的方法观察转移基因的时间-效应曲线。在基因转移3d后以 RT-PCR、Western印迹及免疫组织化学方法检测肾脏AT1的mRNA和蛋白表达与分布,并与正 义(Sen)ODN转移、假注射+肾脏原位电穿孔(Sham)作为对照。结果FITC标记的ODN在基因 转移10 min后主要定位于转移侧肾脏的肾小球,而对侧肾脏FITC荧光阴性。AT1放射自显影定 量分析显示在基因转移后的3 d内,结合有血管紧张素Ⅱ(AngⅡ)的AT1较未转移的对侧肾脏 减少约70％。AT1 AS-ODN基因转移后3 d,转移侧肾脏AT1 mRNA水平较对侧肾脏下降约 43％,蛋白水平下降约67％,而与AT1 Sen-ODN和Sham组转移侧肾脏比较,AT1的mRNA水平 下降分别约47％和51％,蛋白水平下降分别约63％和71％。免疫组化检查发现AT1的阻断以系 膜区的表达减少为主。结论 本研究所用的单侧肾动脉注射+原位电穿孔的基因转移方法成功 地抑制了单侧肾脏的肾小球AT1受体的表达,为研究全身性疾病肾损害中肾脏局部RAS的短 期作用机制提供了更多还原

【Abstract】 Objective To evaluate the local renin-angiotensin system(RAS)suppressive effect of angiotensin type 1 receptor (AT1) antisense (AS) oligodeoxynucleotide (ODN). Methods In male Wistar rats, ATI1 AS-ODN was locally delivered to one kidney through unilateral injection via renal artery followed by electroporation of the injected kidney. FITC-labeled AS-ODN was used to visualize the localization of transferred ODN on frozen section of the transfected kidney. Renal AT1 suppression was determined by radioautography for receptor binding capacity, RT-PCR for mRNA expression, Western blot as well as immunohistochemistry for protein expression. The effects of ASODN were compared with those caused by AT1 sense ODN (Sen-ODN) or procedure alone (Sham). The gene transfer protocol was optimized in terms of voltage for electroporation and duration of transferred AS-ODN. Results FITC-labeled AS-ODN was distributed mainly in glomeruli of transfected kidney, while it was not detectable in contralateral untransfected kidney. AT1 AS-ODN transfection decreased AT1 binding angiotensin Ⅱ by 70% , mRNA level by 43% and protein expression by 67% compared with those in the contralateral kidney 3 days after transfection. Similarly, AT1 mRNA expression was 47% and 51% less, and protein level was 63% and 71% lower compared to those with Sen-ODN transfections and Sham,respectively. Immunohistochemistry showed that reduced AT1 in glomeruli was primarily localized in the mesangium. Conclusions Local transiection of AT1 AS-ODN can effectively inhibit AT1 in transiected glomeruli. This method can be used to study short-term effects of renal RAS.更多还原