人激肽释放酶基因对肾性高血压大鼠主动脉胶原含量的影响及其机制探讨

Effects of Human Tissue kallikrein Gene on Vascular Remodeling in Renovascular Hypertensive Rats

目的观察重组腺相关病毒介导人激肽释放酶基因对肾性高血压大鼠主动脉胶原含量的影响及其可能的机制。方法将人组织型激肽释放酶基因(HK)克隆入重组腺相关病毒载体(rAAV)中,经三质粒共转染方法在 HEK293细胞中包装成 rAAV-HK 病毒。雄性 Wistar 大鼠24只,随机分为假手术和模型组,采用5/6肾切除的方法构建肾性高血压大鼠模型。模型组大鼠再随机分成单纯手术(n=6)、对照组(n=6)和实验组(n=6)。实验组大鼠单次尾静脉注射1×10^(11)pfU 的 rAAV-HK 病毒。分别于模型构建前、后(基因转染前)及基因转染后1～3月测大鼠尾动脉血压。基因转染3月后处死动物,取其心脏、主动脉和肾脏等脏器提取总 RNA 及蛋白质。主动脉组织切片行天狼星红染色,Western Blot 检测主动脉组织缓激肽 B_2和血管紧张素Ⅱ1型受体(AT_1R)蛋白表达水平。结果 HK 基因转染后3月,实验组大鼠与单纯手术和对照组相比血压明显下降[(163±13)vs(217±16)vs(220±13)mmHg,n=6,P<0.01];主动脉中膜内胶原纤维明显减少;主动脉组织缓激肽 B_2受体蛋白表达水平明显上调;AT_1R 蛋白表达水平则明显下调。结论重组腺相关病毒介导人激肽释放酶基因能够明显降低肾性高血压大鼠血压,同时减少主动脉中膜胶原纤维含量,其机制可能与它上调缓激肽 B_2受体和下调 AT_1R 蛋白在主动脉组织的表达水平有关。

Objective To investigate the effects of recombinant adeno associated virus（rAAV） mediated human tissue kallikrein（HK） gene on vascular remodeling in renovascular hypertensive rats. Methods Human kallikrein cDNA was packed in a rAAV-based plasmid vector and rAAV-HK was produced by cotransfection in HEK293 cells. Twenty four male wistar rats were divided into sham operation （n=6） group and model group（n= 18）. The rats in model group underwent laparotomy with 5/6 nephrectomy and were randomly divided into only operation group（n=6）, control group（n=6） and treatment group （n=6）. Single dose rAAV HK（1×10^11 pfU ） via the tail vein. Arterial pressure was measured using tail cuff method. Collagen fibril in aorta was assessed by Sirius red staining. The protein expression of bradykinin B2 and angiotensin Ⅱ type 1 receptor（AT1 R） in aortic tissue were detected by Western blot. Results Three months after gene delivery, blood pressure of 5/6 nephrectomized rats was significantly lower than that of operation alone without treatment and control rats（163±13 vs 217± 16 vs 220±13 mmHg, P〈0.01）. rAAV-HK treatment reduced collagen fibril deposition in aortic middle membranes and up-regulated protein expression of bradykinn B2 receptor associated with down-regulating expression of AT, R. Conclusion These results indicated that rAAV-HK delivery reduced blood pressure and improved vascular remodeling in renovascular hypertensive rats through regulating bradykinin B2 receplor and AT1R in aortic tissue.