磷酸二酯酶5的短发夹RNA对肌细胞纤维化的作用抑制研究  

Inhibition of phosphodiesterase 5 short hairpin RNA on myocyte fibrosis

作　　者：孙洋 李龙虎[1] 胡爽 陈宏[2] 魏冬梅[3] 马天翔[1] SUN Yang;LI Long-hu;HU Shuang;CHEN Hong;WEI Dong-mei;MA Tian-xiang(Department of Cardiology,The First Hospital of Qiqihar City,Qiqihar 161005,Heilongjiang Province,China;Cadre Ward,The First Hospital of Qiqihar City,Qiqihar 161005,Heilongjiang Province,China;Department of Science and Education,The First Hospital of Qiqihar City,Qiqihar 161005,Heilongjiang Province,China)

机构地区：[1]齐齐哈尔市第一医院,心内科,黑龙江齐齐哈尔161005 [2]齐齐哈尔市第一医院干部病房,黑龙江齐齐哈尔161005 [3]齐齐哈尔市第一医院科教部,黑龙江齐齐哈尔161005

出　　处：《中国临床药理学杂志》2021年第2期128-132,共5页The Chinese Journal of Clinical Pharmacology

基　　金：黑龙江省自然科学基金资助项目(H2016096)。

摘　　要：目的通过短发夹RNA(shRNA)延长对磷酸二酯酶5(PDE5)的抑制作用,探索shRNA-PDE5对细胞存活率的影响以及对心肌梗死后心功能及心室重构的作用及其机制。方法(1)细胞实验:分离SD大鼠骨髓间充质干细胞(MSCs),将Ad-shRNA-PDE5和Ad-Null分别感染体外培养的MSCs,嘌呤霉素筛选稳定表达它们的细胞株,分为shPDE5组和空白对照载体(Ad-Null)组,另设正常组。缺口末端标记法染色检测各组缺氧缺糖(OGD)诱导的稳转MSCs细胞凋亡情况。(2)动物实验:通过结扎左冠状动脉前降支方法建立雄性SD大鼠心肌梗死模型。按照体重将大鼠随机分成6组:假手术组、模型组、对照组、第1转染组、第2转染组和第3转染组,在结扎前降支后,立即在心肌梗死区及周边区4个点各注射经不同处理的MSCs细胞。用蛋白质印迹法检测大鼠心肌中转化生长因子-β(TGF-β)和成纤维细胞生长因子2(FGF2)的蛋白表达水平(光密度值);Masson染色检测心肌梗死区及周边区组织的纤维化情况。结果(1)shPDE5组、Ad-Null组和正常组的凋亡细胞数分别为(6.00±1.00),(14.33±1.52)和(5.33±1.52)个,shPDE5组与Ad-Null组比较,凋亡细胞数明显减少,差异有统计学意义(P<0.05)。(2)假手术组、模型组、对照组、第1转染组、第2转染组和第3转染组的FGF2蛋白表达水平分别为0.11±0.02,0.22±0.05,0.27±0.08,0.28±0.04,0.33±0.05和0.50±0.03;这6组的TGF-β蛋白表达水平分别为0.18±0.02,0.91±0.03,0.72±0.01,0.72±0.03,0.48±0.02和0.27±0.09。第2转染组、第3转染组与假手术组、模型组、对照组和第1转染组比较,上述指标的差异均有统计学意义(均P<0.05)。对照组、第1转染组、第2转染组和第3转染组与模型组比较,大鼠梗死周边区心肌组织细胞纤维化程度依次减弱。结论shRNA-PDE5可通过减少细胞凋亡,调节相关因子的表达,抑制心肌细胞纤维化,从而改善心功能。Objective To explore the effect of shRNA-PDE5 on cell viability,cardiac function and ventricular remodeling after myocardial infarction through the inhibition of phosphodiesterase 5(PDE5)by short hairpin RNA(shRNA).Methods(1)Cell experiment:The mesenchymal stem cells(MSCs)of SD rats were isolated,and the MSCs cultured in vitro were infected with Ad-shRNA-PDE5 and Ad-Null,respectively.Puromycin cell lines were screened for stable expression and were divided into shPDE5 group,blank control vector(Ad-Null)group,and normal group was set up.TdT-mediated dUTP nick-end labeling staining was used to detect the apoptosis of stabilized MSCs induced by oxygen and sugar deprivation(OGD)in each group.(2)Animal experiment:Male SD rats were selected and the myocardial infarction model was established by ligating the anterior descending branch of the left coronary artery.Rats were randomly divided into 6 groups:sham operation group,model group,control group,transfection-1 group,transfection-2 group,and transfection-3 group.After descending branches before ligation,MSCs with different treatments were injected at each of 4 points in the myocardial infarction area and surrounding areas immediately after ligation.Western blot was used to detect the expression levels(optical density values)of transforming growth factor-β(TGF-β)and fibroblast growth factor 2(FGF2)in rat myocardium.Masson staining was used to detect fibrosis in myocardial infarction area and surrounding area.Results(1)The number of apoptotic cells in shPDE5 group,Ad-Null group and normal group were(6.00±1.00),(14.33±1.52)and(5.33±1.52),respectively.The number of apoptotic cells in shPDE5 group and Ad-Null group was significantly reduced,and the difference was statistically significant(P<0.05).(2)The expression of FGF2 protein in the sham operation group,model group,control group,transfection-1 group,transfection-2 group,and transfection-3 group were 0.11±0.02,0.22±0.05,0.27±0.08,0.28±0.04,0.33±0.05 and 0.50±0.03,respectively;the expression of TGF-βprotein i

关 键 词：短发夹RNA 磷酸二酯酶5 骨髓间充质干细胞 心肌细胞 纤维化 

分 类 号：R97[医药卫生—药品]