RNAi基因沉默作用于肾性高血压大鼠的实验研究  

The Effects of Recombinant Adeno-Associated Virus Vector Delivery of Angiotensin Ⅱ Type 1 Receptor shRNA on Blood Pressure and Angiotensin Ⅱ Type 1 Receptor mRNA Expression in Renal Hypertensive Rats

作　　者：陈闽荔[1] 张金莲[1] 陈树涛[1] 

机构地区：[1]天津市胸科医院心内科,天津300051

出　　处：《中华高血压杂志》2010年第1期65-70,共6页Chinese Journal of Hypertension

摘　　要：背景高血压是多基因遗传病,通过调节体内基因表达而控制高血压的基因疗法是一种长期有效的解决高血压的最终临床方案。目的观察应用RNAi技术靶向基因沉默血管紧张素1型受体(AT1R)对肾性高血压大鼠血压及组织AT1R基因表达的影响。方法健康雄性Sprague-Dawley(SD)大鼠随机分为实验组(n=12)、高血压对照组(n=12)和正常对照组(n=12),采用左肾动脉狭窄法建立两肾一夹高血压大鼠模型,实验组经鼠尾静脉单次注射感染性滴度为1.5×109的携带AT1RshRNA重组腺相关病毒载体(rAAV-AT1R-shRNA),高血压对照组和正常对照组单次注射重组复制缺陷型腺病毒(rAAV-EGFP),感染性滴度为2.9×109。注射前及注射后每天定时监测血压及心率,于血压出现明显下降时处死部分动物,用放射免疫法测定血浆血管紧张素Ⅱ(AngⅡ)的含量,在荧光显微镜下观察rAAV-AT1R-shRNA在体内重要脏器组织的分布情况,采用荧光定量PCR检测心脏、肾脏及主动脉组织AT1RmRNA的表达情况。结果注射rAAV-AT1R-shRNA24h后,收缩压较注射前明显下降[(22.3±5.5)mmHg,P<0.01],最大降压幅度可达30.2mmHg,与高血压对照组比较差异有统计学意义(P<0.05),降压作用可持续7d;高血压对照组及正常对照组较注射前血压均未见明显变化;3组动物的心率变化不明显;正常对照组血浆AngⅡ浓度[(50.5±5.3)ng/L]明显低于实验组[(78.6±11.2)ng/L]和高血压对照组[(82.1±8.5)ng/L,P<0.05],而实验组与高血压对照组比较无统计学意义(P>0.05)。肾脏、心脏、肝脏、主动脉及肾上腺组织在荧光显微镜下可见大量荧光表达,而脑干组织未见荧光表达;实验组心脏、肾脏及主动脉AT1R的mRNA表达量明显低于高血压对照组(P<0.01)。结论经静脉注射后rAAV-AT1R-shRNA可被重要脏器吸收,在mRNA水平抑制AT1R的基因表达,对肾性高血压大鼠可起到明显且持久的降压作用,但对循环中的AngⅡ浓度影响不明显�Background Hypertension is a multigenetic inheritable disease.Gene therapy by regulating gene expression showed long term effects and less side effects,and has been emerged to be a potential and prospective treatment.Objective To investigate the effects of retroviraladeno-associated virus vector containing shRNA targeted on the AT1R gene （rAAV-AT1R-shRNA） on blood preesure（BP） in renal hypertensive rats,and the inhibitory effect of RNA interference （RNAi） on AT1R mRNA expression in renal hypertensive rats.Methods Two-kideny one-clip （2K1C） renal hypertension （RH） were established in SD rats and randomly to receive rAAV-AT1R-shRNA,1.5×10^9 particles/mL（n=12,ip）,as treated group or retroviral vector （rAAV-EGFP）,2.9×10^9 particles/mL,ip,as vehicle group,normal SD rats served as controls （n=12）.SBP was measured before and after treatment.Animals were euthanized and blood,brain,heart,liver,kidney,aorta and adrenal gland were collected to identify the sites of rAAV-AT1R-shRNA expression by fluorescence microscope.Angiotensin Ⅱwas assessed by radioimmunology.Results 24 hours after single injection of rAAV-AT1R-shRNA,SBP was reduced by （22.3±5.5）mmHg compared to before intervention （P〈0.01）,and controls（P〈0.05）with the antihypertensive effect lasting for 7 days,while no BP changes were found in control and vehicle rats.Plasma angiotensin Ⅱ concentration in treated rats（78.6 ±11.2）ng/L were significantly higher compared to control rats （50.5±5.3） ng/L with no differences between treated and vehicle rats,as well as between treated rats and hypertension control rats（82.1 ±8.5）ng/L.Marked expression of AT1R-shRNA was demonstrated in heart,liver,kidney,aorta and adrenal gland,except brain.Furthermore,AT1R mRNA levels in heart,kidney and aorta were significantly decreased in the rAAV-AT1R-shRNA treated rats compared with vehicle treated rats（P〈0.01）.Conclusion The results indicate that exogenous rAAV-AT1R-shRNA could be expressed in many main organs an

关 键 词：RNAI 肾性高血压 血管紧张素Ⅱ 血管紧张素1型受体 

分 类 号：R544.1[医药卫生—心血管疾病]