骨髓间充质干细胞来源的微颗粒对心肌肥厚的作用及其机制

Effect of microparticles derived from bone marrow mesenchymal stem cells on cardiomyocyte hypertrophy and its mechanism

目的 探讨骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)来源的微颗粒(microparticles,MPs)对心肌肥厚的作用及其机制。方法 对间充质干细胞(MSCs)进行成骨成脂诱导分化,经分离纯化后,透射电镜观察形态特征,流式细胞术鉴定MPs表面抗原。利用异丙肾上腺素(isoprenaline,ISO)诱发建立大鼠心肌肥厚模型,超声心动图检测大鼠心脏结构及功能后,进行心脏及分离的左心室的各项指标检测。将急性分离的大鼠单个心肌细胞分为对照(Control)、心肌细胞肥大(ISO)、MPs、MPs上清液(Supernatant)组,qRT-PCR法检测心肌细胞心房利钠肽(atrial natriuretic peptide,ANP)、脑利钠肽(B-type natriuretic peptide,BNP)mRNA表达量;ELISA法检测心肌细胞钙调蛋白依赖性蛋白激酶Ⅱ(calmodulin-dependent protein kinaseⅡ,CaMKⅡ)、磷酸化钙调蛋白依赖性蛋白激酶Ⅱ(phosphorylated calmodulin-dependent protein kinaseⅡ,p-CaMKⅡ)表达量;全细胞膜片钳记录各组大鼠单个心肌细胞L-型钙电流(LCa-L)。结果 MSCs成骨分化骨结节经茜素红染色后变成红色,成脂分化脂滴经油红O染色后变成红色;透射电镜下可见MPs膜结构完整,轮廓清晰,直径约200 nm;MPs表面标记物CD29和CD90表达阳性率分别为(98.24±0.82)%和(97.69±1.83)%。与Control组相比,ISO组大鼠左心室舒张末期内径(left ventricular end diastolic dimension,LVEDD)明显减少(t=5.065,P <0.05),室间隔舒张末期厚度(interventricular septum end-diastolic dimension,IVSd)、左心室后壁舒张末期厚度(left ventricular posterior wall dimension,LVPWd)、心重-体重比(heart weight to body weight ratio,HW/BW)、心重-胫骨长度比(heart weight to tibial length ratio,HW/Tibia)、左心重-胫骨长度比(left heart weight to tibial length ratio,LV/Tibia)明显升高(t分别为4.013、2.368、4.392、5.043、6.120,P均<0.05),建模成功;ISO组大鼠肥大心肌细胞ANP、BNP mRNA表达量均较Control组明显升高(t分别为25.120、18.261,P均<0.01);经48μg/mL MPs孵育48 h后的MPs组ANP、BNP mRNA表达量较ISO组显著下降(t分别为12.110、3.526,P均<0.05);ISO组CaMKⅡ和p-CaMKⅡ蛋白表达水平较Control组显著升高(t分别为3.278、4.181,P均<0.05),MPs组p-CaMKⅡ蛋白表达水平显著下降(t=5.420,P <0.05);ISO组钙电流密度较Control组明显增高(t=15.261,P <0.01),MPs组较ISO组明显降低(t=6.216,P <0.05)。结论 MSC-MPs可显著抑制ISO诱导的大鼠心肌细胞肥大,这一效应与其下调心肌细胞CaMKⅡ、抑制L-型钙通道有关。

Objective To investigate the effect of microparticles(MPs)derived from bone marrow mesenchymal stem cells(BMSCs)on myocardial hypertrophy and its mechanism.Methods The osteogenic differentiation and adipogenic differentiation of mesenchymal stem cells(MSCs)were induced.After isolation and purification,the morphological characteristics were observed by transmission electron microscope,and the MPs surface antigen was identified by flow cytometry.Myocardial hypertrophy model was induced by using isoprenaline(ISO)in rats,which were measured for the cardiac structure and function by echocardiography,and then detected for various indexes of the heart and isolated left ventricle.Single ventricular myocytes of rats were acutely isolated and divided into control group(Control group),cardiomyocyte hypertrophy group(ISO group),MPs group(MPs group),and MPs supernatant group(Supernatant group).The mRNA expressions of atrial natriuretic peptide(ANP)and B-type natriuretic peptide(BNP)were detected by qRTPCR.The expression levels of calmodulin-dependent protein kinaseⅡ(CaMKⅡ)and phosphorylated calmodulin-dependent protein kinaseⅡ(p-CaMKⅡ)were detected by ELISA.The L-type calcium current(LCa-L)in single ventricular myocyte of various groups was recorded by whole-cell patch clamp.Results The bone nodules of MSCs osteogenic differentiation turned red after alizarin red staining,and lipid droplets of adipogenic differentiation turned red after oil red O staining;Under transmission electron microscope,MPs membrane had a complete structure,a clear outline and a diameter of about 200 nm;The positive rates of CD29 and CD90 on the surface of MPs were(98.24±0.82)%and(97.69±1.83)%,respectively.Compared with Control group,the left ventricular end diastolic dimension(LVEDD)reduced signifi-cantly(t=5.065,P<0.05),while the interventricular septum end-diastolic dimension(IVSd),left ventricular posterior wall dimension(LVPWd),heart weight to body weight ratio(HW/BW),and heart weight to tibial length ratio(HW/Tibia)significantly increased in ISO group(t=4.013,2.368,4.392,5.043 and 6.120,respectively,each P<0.05),indicating that the hypertrophic model was successfully established.The expression levels of ANP and BNP mRNA in hypertrophic cardiomyocytes of rats in ISO group were significantly higher than those in Control group(t=25.120 and 18.261,respectively,each P<0.01);While the expression levels of ANP and BNP mRNA in MPs group significantly reduced after incubation with 48μg/mL MPs for 48 h compared with ISO group(t=12.110 and 3.526,respectively,each P<0.05);The expression levels of CaMKⅡand p-CaMKⅡin ISO group were significantly higher than those in Control group(t=3.278 and 4.181,respectively,each P<0.05),while the expression of p-CaMKⅡin MPs group decreased significantly(t=5.420,P<0.05);The calcium current density in ISO group was significantly higher than that in Control group(t=15.261,P<0.01),while that in MPs group was significantly lower than that in ISO group(t=6.216,P<0.05).Conclusion MSC-MPs can significantly inhibit ISO-induced cardiomyocyte hypertrophy in rats,which is related to its down-regulation of cardiomyocyte CaMKⅡand inhibition of L-type calcium channel.