靶向磷酸二酯酶5的短发夹RNA导入后的间充质干细胞对心肌梗死后心室重构的影响

Effects of MSCs with shRNA targeting phosphodiesterase 5 on ventricular remodeling after myocardial infarction

目的探讨磷酸二酯酶5(PDE5)短发夹RNA(shRNA)重组腺病毒载体[PDE5shRNA]导入后的间充质干细胞(MSCs)对心肌梗死(MI)后心室重构的影响。方法构建PDE5shRNA转染的MSCs,结扎大鼠冠状动脉左前降支(LAD)建立MI模型,随机分成假手术组(n=4,只穿线不结扎LAD);模型组(n=4,穿线并结扎LAD建立MI模型);MSCs-Ad-Null组(n=4,MI模型基础上注射经空白腺病毒载体转染的MSCs);MSCs-shRNA-PDE5组(n=4,MI模型基础上注射经PDE5shRNA转染的MSCs)。建模后,每组各取1只大鼠取心脏进行病理检验,用于判断造模成功,余大鼠入组观察相关指标。4周后心脏超声检测各组大鼠心功能指标[左室收缩末期内径(LVESd)、左室舒张末期内径(LVEDd)、左室射血分数(LVEF)和左室短轴缩短率(FS)]。HE染色观察心肌病理改变,Masson染色观察心肌纤维化情况,qRT-PCR方法检测大鼠心肌中PDE5、环磷酸鸟苷(cGMP)和蛋白激酶(PKG)mRNA的相对表达量。结果实验4周后,(1)心脏超声示:MI各组大鼠心室腔明显扩张,左心室心肌收缩功能明显降低。与假手术组相比,MI各组大鼠的LVESd、LVEDd增大,LVEF、FS降低(P均<0.05);与模型组相比,MSCs-Ad-Null组、MSCs-shRNA-PDE5组的LVESd、LVEDd降低,MSCs-shRNA-PDE5组的LVEF、FS升高(P均<0.05)。(2)心肌病理结果示:与模型组比较,MSCs-Ad-Null组、MSCs-shRNA-PDE5组的心肌病理改变逐渐减轻。(3)qRT-PCR法结果示:与假手术组相比,模型组、MSCs-Ad-Null组PDE5 mRNA表达升高、cGMP和PKG mRNA表达降低(P均<0.05);按模型组→MSCs-Ad-Null组→MSCs-shRNA-PDE5组之序,PDE5 mRNA表达递降,cGMP、PKG mRNA表达递升(P<0.05,P<0.01)。结论PDE5shRNA导入后的MSCs可改善MI后心功能,减轻心肌病理改变及心肌纤维化,同时通过调节PDE5、cGMP、PKG的表达,发挥抑制心室重构的作用。

Objective To investigate the effects of mesenchymal stem cells(MSCs)with short hairpin RNA(shRNA)targeting phosphodiesterase 5(PDE5)on ventricular remodeling after myocardial infarction(MI).Methods MI model rats were established by ligating left anterior descending coronary artery(LAD),and MSCs were constructed by transfecting with PDE5 shRNA.The rats were randomly divided into sham operation group(n=4,only threading without ligating LAD),the model group(n=4,threading and ligating LAD),MSCs-Ad-Null group(n=4,injected with MSCs transfected with blank adenovirus vector based on MI model)and MSCs-shRNA-PDE5 group(n=4,injected with MSCs transfected with PDE5shRNA based on MI model).After modeling,one rat heart from each group was taken for pathological examination to judge the success of modeling.The relevant indicators were observed in other rats.After 4 weeks,cardiac function indexes were measured by echocardiography,including left ventricular end-systolic diameter(LVESd),left ventricular end-distolic diameter(LVEDd),left ventricular ejection fraction(LVEF)and percent fractional shortening(FS)of left ventricular short axis;myocardial pathological changes were observed by HE staining;myocardial fibrosis was observed by Masson staining;the relative expression level of PDE5,cyclic guanosine monophosphate(cGMP)and protein kinase G(PKG)mRNA were detected by real-time quantitative polymerase chain reaction(qRT-PCR).Results After 4 weeks,echocardiography showed the obviously dilated ventricle cavity and decreased myocardial systolic function of left ventricle in MI rats.Compared with sham operation group,LVESd and LVEDd increased,and LVEF and FS decreased in other three groups(all P<0.05).Compared with model group,LVESd and LVEDd decreased in MSCs-Ad-Null group and MSCs-shRNA-PDE5 group,and LVEF and FS increased in MSCs-shRNA-PDE5 group(all P<0.05).Myocardial pathological results showed that the myocardial pathological changes were alleviated gradually in MSCs-Ad-Null group and MSCs-shRNA-PDE5 group.qRT-PCR results showed that compared with sham operation group,the expression of PDE5 mRNA increased,and the expressions of cGMP and PKG mRNA decreased in model group and MSCs-Ad-Null group(all P<0.05).According to the order of model group,MSCs-Ad-Null group and MSCs-shRNA-PDE5 group,the expression of PDE5 mRNA decreased gradually,and the expressions of cGMP and PKG mRNA increased gradually(P<0.05,P<0.01).Conclusion MSCs with PDE5shRNA can improve cardiac function,alleviate myocardial pathological changes and myocardial fibrosis after MI and inhibit ventricular remodeling by regulating the expressions of PDE5,cGMP and PKG.