重组腺病毒介导Klotho基因转导对大鼠心力衰竭心肌重构的影响

Impact of transfection with recombinant adenovirus vector-mediated Klotho gene on myocardial remodeling in a rat model of heart failure

目的 探讨重组腺病毒介导的Klotho基因转染对大鼠心力衰竭（心衰）进程以及心肌重构的影响,并探讨其机制.方法 SPF级SD大鼠,随机数表法分为5组,即正常对照组（对照组,未给予任何干预）,心衰组（只做心衰模型,不给予其他干预）,生理盐水组（在心衰模型的基础上尾静脉注射生理盐水,0.5 ml/只）,腺病毒载体介导的增强荧光蛋白组（Ad.EGFP组,在心衰模型的基础上用腺病毒载体介导增强荧光蛋白,2×1010 pfu,0.5 ml/只）,以及腺病毒载体介导的目的基因组（Ad.Klotho组,在心衰模型的基础上用腺病毒载体介导目的基因,2×1010pfu,0.5 ml/只）,每组5只.异丙肾上腺素连续腹腔注射法建立大鼠心衰模型.构建重组腺病毒载体pDC316-CMV-EGFP-rKlotho,标记为Ad.Klotho,并将其转导至相应的心衰大鼠模型.建模第6周,采用超声心动图测定各组大鼠的左心室射血分数（LVEF）;多道生理记录仪测定各组大鼠血液动力学变化;检测各组大鼠心肌组织病理学改变;冰冻切片后镜下观察各组大鼠心肌组织荧光蛋白表达水平;实时荧光定量聚合酶链反应评估各组大鼠心肌组织中Klotho基因的表达水平,以及心肌纤维化相关因子Ⅰ型胶原和Ⅲ型胶原的表达水平;ELISA法检测各组大鼠血浆中B型利钠肽（BNP）的表达水平.结果 心衰组、生理盐水组、Ad.EGFP组和Ad.Klotho组大鼠心肌组织苏木素伊红染色均可见心肌细胞断裂、心肌间质增生、心肌细胞肥大和结构改变.对照组大鼠心肌组织病理切片未见上述改变.通过比较发现,Ad.Klotho组大鼠心肌纤维化程度、重构程度以及心肌细胞肥大程度均较心衰组、生理盐水组和Ad.EGFP组低.建模第6周,Ad.Klotho组和Ad.EGFP组大鼠心肌组织中仍可见EGFP绿色荧光表达,而对照组、心衰组和生理盐水组则未见.各组大鼠心肌组织中Klotho、胶原Ⅰ a1和Ⅲa1 mRNA均有表达.其中,对照组和Ad.Klotho组Klotho mRNA表达水平较高,与其他3组比较差异均有统计学意义（P均＜0.05）,而心衰组、生理盐水组和Ad.EGFP组间Klotho mRNA表达水平差异无统计学意义.心衰组、生理盐水组和Ad.EGFP组胶原Ⅰ a1、Ⅲa1 mRNA表达水平均较对照组和Ad.Klotho组高（P均＜ 0.05）,且三组间比较差异无统计学意义.超声心动图结果示心衰组、生理盐水组、Ad.EGFP组和Ad.Klotho组大鼠总体LVEF为（42.27±3.22）％低于对照组的（72.13±2.97）％（P＜0.01）.对照组和Ad.Klotho组心率均高于心衰组、生理盐水组和Ad.EGFP组（P均＜0.05）.对照组和Ad.Klotho组左心室内压均高于心衰组、生理盐水组和Ad.EGFP组（P均＜0.01）.对照组和Ad.Klotho组左心室舒张末压均低于心衰组、生理盐水组和Ad.EGFP组（P均＜0.01）.对照组和Ad.Klotho组左心室内压最大上升速率（＋dp/dtmax）均大于心衰组、生理盐水组和Ad.EGFP组（P均＜0.05）.对照组和Ad.Klotho组左心室内压最大下降速率（-dp/dtmax）亦均大于心衰组、生理盐水组和Ad.EGFP组（P均＜0.05）.心衰组、生理盐水组、Ad.EGFP组和Ad.Klotho组大鼠心脏体质量指数分别为（6.50±0.32）、（6.46±0.18）、（6.34±0.15）和（5.23±0.19） g×l 000/g均明显高于对照组（2.84±0.25）g×1 000/g,P均＜0.05,但Ad.Klotho组又明显低于心衰组、生理盐水组和Ad.EGFP组（P＜0.05）,心衰组、生理盐水组和Ad.EGFP组间差异则无统计学意义.心衰组、生理盐水组、Ad.EGFP组和Ad.Klotho组大鼠血清中BNP水平均明显高于对照组（P均＜0.01）,但Ad.Klotho组明显低于心衰组、生理盐水组和Ad.EGFP组（P均＜0.05）.结论 腹腔注射异丙肾上腺素可成功建立大鼠心衰模型.Klotho基因可抑制心肌纤维化、心肌肥大及胶原纤维增生.

Objective To assess the impact of transfection with recombinant adenovirus vector-mediated Klotho gene on myocardial remodeling in a rat model of heart failure （HF） by intraperitoneal injection of isoproterenol.Methods Rats were divided into 5 groups by table of exponential random numbers：normal control group,HF group,saline-control HF group,recombinant adenovirus vector transfection group （Ad.EGFP group,2 × 1010 pfu,0.5 ml/rat）,pDC316-CMV-EGFP-rKlotho transfection group （Ad.Klotho group,n =5 each）.Left ventricular ejection fraction （LVEF） was obtained by echocardiography,hemodynamic parameters obtained by multi-channel physiological recorder,myocardial tissue underwent pathohistological examination.Additionally,the green fluorescin expression was observed on frozen heart section.Myocardial fibrosis correlated gene expression including Klotho gene,collagen Ⅰ and Ⅲ was detected by real time-PCR.Moreover,plasma levels of B-type natriuretic peptide （BNP） were measured with ELISA.Results Compared to saline control HF group,LVEF,LVSP and ± dp/dtmax were significantly increased,myocardial fibrosis and myocardial remodeling were significantly attenuated in the Ad.Klotho group and there was green fluorescin distribution in myocardial tissues of Ad.Klotho group.Klotho expression was down-regulated and collagen Ⅰ and Ⅲ expression was upregulated in HF rats compared to normal control group （all P ＜ 0.05） and these changes could be significantly reversed in Ad.Klotho group （all P ＜ 0.05）.Plasma BNP level was also significantly lower in Ad.Klotho group than in HF group （P ＜ 0.05）.Conclusions Klotho gene transfection could improve cardiac function and attenuate cardiac remodeling and reducing myocardial fibrosis.