中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (35): 5626-5631.doi: 10.12307/2021.291

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

靶向血管紧张素原RNA干扰对自发性高血压模型大鼠的影响

袁丽粉1，曹  爽2，孙  婷3   

  1. 上海交通大学医学院附属第九人民医院，1呼吸与危重症医学科，2麻醉科，3心血管内科，上海市   200011
  • 收稿日期:2020-12-14 修回日期:2020-12-18 接受日期:2021-01-30 出版日期:2021-12-18 发布日期:2021-08-03
  • 通讯作者: 孙婷，博士，主任医师，上海交通大学医学院附属第九人民医院心血管内科，上海市 200011
  • 作者简介:袁丽粉，女，1985年生，山东省聊城市人，汉族，2012年上海交通大学医学院毕业，硕士，主治医师，主要从事高血压的研究
  • 基金资助:
    国家自然科学基金面上项目(81770505) ，项目负责人：孙婷

Effect of angiotensinogen-targeted RNA interference in spontaneously hypertensive rats

Yuan Lifen1, Cao Shuang2, Sun Ting3   

  1. 1Department of Respiratory Medicine and Critical Care Medicine, 2Department of Anesthesiology, 3Department of Cardiovascular Medicine, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China
  • Received:2020-12-14 Revised:2020-12-18 Accepted:2021-01-30 Online:2021-12-18 Published:2021-08-03
  • Contact: Sun Ting, MD, Chief physician, Department of Cardiovascular Medicine, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China
  • About author:Yuan Lifen, Master, Attending physician, Department of Respiratory Medicine and Critical Care Medicine, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China
  • Supported by:
    the National Natural Science Foundation of China (General Program), No. 81770505 (to ST)

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摘要：

文题释义：
RNA干扰(RNA interference，RNAi)：是指在进化过程中高度保守的、由双链RNA诱发的、同源mRNA高效特异性降解的现象。RNAi是一项新兴的基因阻断技术，是一种由双链RNA介导的简单、快速沉默生物体内基因表达的方法，它将与mRNA相对应的正义RNA和反义RNA组成的双链RNA导入细胞内，可以使得mRNA发生特异性的降解，从而特异性剔除或关闭特定基因的表达。
自发性高血压大鼠(spontaneously hypertensive rats，SHR)：是用一只收缩压持续在150-175 mmHg的雄性Wistar京都种大鼠与收缩压为  130-140 mmHg的雌性Wistar京都种大鼠交配，得到收缩压都大于150 mmHg的子代，再选用血压较高的大鼠进行近亲交配，依次进行这种选择性近亲交配20代而获得稳定的高血压遗传性，从而建立了SHR品种，实验中应用的SHR大鼠为直接购买模型。
WKY大鼠：维持正常血压的Wistar大鼠被称为京都种Wistar大鼠(Wistar Kyoto rats)，简称WKR。在实验中做对照组。
GPE-AGTshRNA纳米复合物：由GPE基因输送载体携带血管紧张素原(AGT)基因制备而成。  
背景：前期研究验证了GPE-AGTshRNA纳米复合物在体外对肝细胞不仅有较好的转染效率、较低的细胞毒性，而且在基因和蛋白水平上明显降低了血管紧张素原的表达。
目的：观察靶向血管紧张素原 RNA干扰对自发性高血压大鼠血压及其主要靶器官心脏的影响，以期寻找一种新的降压策略。
方法：实验分为4组：自发性高血压大鼠随机均分为基因治疗组、阴性对照组、空白对照组；同时设立正常血压对照大鼠为WKY组。实验共计9个周期，10 d为1个周期。于每1个周期的第1天经鼠尾静脉注射给药：WKY组注射无菌注射用水500 μL，基因治疗组注射GPE-AGT shRNA纳米复合物500 μL，阴性对照组注射GPE-NC shRNA纳米复合物500 μL，空白对照组注射无菌注射用水500 μL；②Real-time PCR法检测肝脏血管紧张素原 mRNA的表达，Western blot法检测肝脏血管紧张素原蛋白的表达，ELISA法测定血清血管紧张素原和血管紧张素Ⅱ的水平，尾袖法测量大鼠尾动脉收缩压，心超检测大鼠心室功能，光镜观察心肌组织结构病变。
结果与结论：①基因治疗组大鼠肝脏血管紧张素原 mRNA和蛋白质表达以及血清血管紧张素原、血管紧张素Ⅱ水平与阴性对照组、空白对照组相比明显下降(P < 0.01)；②首次注射后第3天，基因治疗组自发性高血压大鼠尾动脉压显著下降(28±4) mmHg，与治疗前比较有显著性差异(P < 0.01)；③基因治疗组左心室射血分数、左室短轴缩短率明显升高，左室后壁厚度明显下降，而且光镜下心肌细胞肥大明显减轻；④结果说明，靶向血管紧张素原 RNA干扰有效下调了肝脏血管紧张素原基因的表达，控制血压的同时显著改善了心肌肥大及心室功能。
https://orcid.org/0000-0001-9464-2386 (袁丽粉) 

关键词: 高血压, 血管紧张素原, RNA干扰, 自发性高血压大鼠, 基因治疗, 心肌肥大

Abstract: BACKGROUND: Recent studies have found that GPE-AGTshRNA nanocomposite cannot only have better transfection efficiency and lower cytotoxicity to hepatocytes in vitro, and also significantly reduce the expression of angiotensinogen at the gene and protein levels.
OBJECTIVE: To observe the effects of angiotensinogen-targeted RNA interference on blood pressure and cardiac protection in spontaneously hypertensive rats, attempting to find a new antihypertensive strategy.
METHODS: Spontaneously hypertensive rats were randomly evenly divided into gene therapy group, negative control group and blank control group, and normal Wistar Kyoto rats (WKY) were set as normal blood pressure control group (WKY group). There were nine sessions in total, with 10 days as one session. On the 1st day of each session, administration in each group was given via the rat tail vein: 500 μL of sterile water for injection in the WKY group, 500 μL of GPE-AGTshRNA nanocomplex in the gene therapy group, and 500 μL of GPE-NC shRNA nanocomplex in the negative control group, and 500 μL of sterile water in the blank control group. We used real-time PCR and western blot assay respectively to detect hepatic angiotensinogen mRNA and protein levels. The serum levels of angiotensinogen and angiotensin II were measured by ELISA. Systolic blood pressure of the tail artery was measured with tail cuff method. Ventricular function was detected by echocardiography. Myocardial lesion was observed under light microscopy.
RESULTS AND CONCLUSION: Angiotensinogen mRNA and protein expression and serum angiotensinogen and angiotensin II levels were significantly lower in the gene therapy group than the negative and blank control groups (P < 0.01). On the 3rd day after the first injection, tail arterial pressure of rats decreased significantly by (28±4) mmHg in the gene therapy group, which was significantly different from that before treatment (P < 0.01). The parameters of left ventricular ejection fraction and left ventricular fractional shortening were significantly increased in the gene therapy group, while the thickness of the left ventricular posterior wall was significantly reduced, and cardiomyocyte hypertrophy were significantly relieved under light microscope. To conclude, angiotensinogen-targeted RNA interference can effectively control blood pressure through downregulation of angiotensinogen, and meanwhile significantly  alleviate myocardial hypertrophy and improve ventricular function.

Key words: hypertension, angiotensinogen, RNA interference, spontaneously hypertensive rats, gene therapy, myocardial hypertrophy

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