血管紧张素Ⅱ-1型受体的shRNA对自发性高血压大鼠血压影响的实验研究

The effects of shRNA targeting angioteusin Ⅱ type 1 receptor on blood pressure and AT1R mRNA expression in spontaneously hypertensive rats

目的观察以重组腺病毒为载体的血管紧张素Ⅱ-1型受体的shRNA（AdS—AT1R—shRNA）对自发性高血压大鼠（SHR）血压的影响及对组织血管紧张素Ⅱ-1型受体（AT1R）基因表达的影响。方法在293细胞内扩增已构建好的荧光蛋白标记的携带AT1R shRNA的重组腺病毒（AdS—AT1R—shRNA），TCID50法测定重组腺病毒滴度。22只SHR随机分为2组，实验组（n=11）和高血压对照组（n=11），另设11只Wistar—Kyoto（WKY）大鼠为正常血压对照组，实验组SHR经鼠尾静脉单次注射Ad5—AT1R—shRNA，Ad5—AT1R—shRNA经TCID50法测定感染性滴度为1．7×10^9TCID50／ml，高血压对照组和正常血压对照组经鼠尾静脉单次注射对照重组复制缺陷型腺病毒（Ad5—EGFP），感染性滴度为7.9×10^9TCID50／ml。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，取出心脏、肝脏、肾脏、主动脉及肾上腺组织，在荧光显微镜下观察他们对Ad5—AT1R—shRNA的吸收情况，采用荧光定量PCR检测肝脏、肾脏及主动脉组织AT1R mRNA的表达情况。结果实验开始24h后，实验组收缩压[（163±7）mmHg，1mmHg=0．133kPa]出现明显下降，最大降压幅度达29mmHg，与SHR组[（182±8）mmHg]比较差异有统计学意义（P〈0．05），此后降压作用可持续5天，最长可持续7天。SHR组和WKY组血压均未见明显下降，SHR组有的血压可见继续升高。3组动物的心率变化不明显，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。实验组肾脏及主动脉AT1R的mRNA表达量（分别为0．086±0．014，0，051±0．023）明显低于SHR组（分别为0．362±0．042，0．463±0．045），P〈0，01。结论AdS—AT1R—shRNA经静脉注射后可被许多重要脏器吸收，且对SHR的AT1R起到RNA干扰的作用，在mRNA水平抑制AT1R的基因表达。AdS—AT1R·shRNA通过阻抑AT1R生成对SHR起到明显且持久的降压作用。

Objective To investigate the effects of retroviral vector containing shRNA targeting rat angiotensin Ⅱ type 1 receptor （AT1R） gene （Ad5-AT1R-shRNA） on blood pressure and AT1R mRNA expression in spontaneously hypertensive rats （ SHR ）. Methods Retroviral vector containing shRNA targeting rat AT1R gene was constructed and propagated further in 293 cells. SHR rats were randomly divided into SHR ± Ad5-AT1R-shRNA （1.7 × 10^9 TCID50/ml） group and SHR （Ad5-EGFP, 7.9 × 10^9 TCID50/ml, n= 11 each） and 11 male Wistar-Kyoto rats （WKY） serve as normal controls （Ad5-EGFP, 7.9 × 109 TCID50/ml）. Systolic blood pressure was measured before and after single intravenous injection of Ad5-AT1R-shRNA or Ad5-EGFP. Heart, liver, kidney, aorta and adrenal gland were removed after blood pressure measurement. Tissue Ad5-AT1R-shRNA expression was detected with fluorescence microscope and AT1R mRNA in liver, kidney and aorta was measured by fluorescence quantitative PCR. Results Ad5-AT1R-shRNA significantly reduced blood pressure compared with controls（ -29 mm Hg, 1 mm Hg = 0. 133 kPa, P 〈 0. 05 ） 24 hours after single injection and this antihypertensive effect could last for 5 to 7 days. Ad5-AT1R-shRNA expression detected with fluorescence microscope was significantly increased in heart, liver, kidney, aorta and adrenal gland post Ad5-AT1R-shRNA injection. AT1R mRNA in kidney and aorta （0. 086 ± 0. 014,0. 051 ± 0. 023 ） were significantly decreased in Ad5-AT1R-shRNA treated rats compared with SHR control rats （ 0. 362 ± 0. 042,0. 463 ± 0. 045, P 〈 0. 01 ）. Conclusion The results indicate that Ad5-AT1R-shRNA could inhibit the tissue AT1R mRNA expression and produce prolonged antihypertensive effects in SHR rats.