CAJ | 학술논문

目的探讨微小RNA(miRNA)-199a在心肌肥厚中的作用.方法 (1)Sprague-Dawley (SD)大鼠12只,分为腹主动脉缩窄(abdominal aortic constriction,AAC)组(AAC组,n=6)和假手术组(n=6).AAC组通过腹主动脉缩窄术建立大鼠心肌肥厚模型.实时定量聚合酶链反应(qRT-PCR)检测心肌中部分miRNA表达的变化.(2)SD乳鼠心肌细胞分为两组,即miRNA-199a重组腺病毒(Ad-miRNA-199a)组(n=8)和腺病毒载体(Ad-vector)组(n=8),分别转染SD乳鼠心肌细胞48 h后qRT-PCR检测心肌细胞中miRNA-199a以及心肌肥厚标志分子α肌球蛋白重链(α-myosin heavy chain,αMHC)、β肌球蛋白重链(β-myosin heavy chain,βMHC)、心房钠尿肽(atrial natriuretic peptide,ANP)编码基因myh6、myh7、Nppa的表达变化,并利用免疫荧光分析检测细胞表面积的变化.(3)SD乳鼠心肌细胞分为两组,即miRNA-199a的反义寡核苷酸(As-miRNA-199a)组(n=8)和混杂寡核苷酸(As-ctl)组(n=8),分别转染SD乳鼠心肌细胞48 h后qRT-PCR检测心肌细胞中miRNA-199a的表达变化.(4)SD乳鼠心肌细胞分为4组,即空白对照组(n=8)、苯肾上腺素组(phenylephrine,PE)(n=8)、PE+As-cd组(n=8)和PE+As-miRNA-199a绀(n=8),分别转染SD乳鼠心肌细胞48 h,qRT-PCR检测心肌肥厚标志分子编码基因的表达变化,免疫荧光检测细胞表面积的变化.结果 (1)qRT-PCR结果显示,AAC组大鼠造模后1周miRNA-1、miRNA-133、miRNA-181a及miRNA-499的表达均显著低于假手术组,而miRNA-199a表达显著高于假手术组.(2)qRT-PCR结果显示,AdmiRNA-199a组SD乳鼠心肌细胞中miRNA-199a的表达显著高于Ad-vector组,myh7的表达亦显著高于Ad-vector组,而myh6的表达低于Ad-vector组.免疫荧光显示Ad-miRNA-199a组SD乳鼠心肌细胞的表面积大于Ad-vector组,P<0.05.(3)qRT-PCR结果显示,As-miRNA-199a组SD乳鼠心肌细胞中miRNA-199a的表达显著低于As-ctl组,P<0.05.(4)qRT-PCR结果显示,PE组Nppa及myh7的表达量显著高于空白对照组,而myh6的表达量低于空白对照组,P<0.05.免疫荧光检测显示,PE+As-miRNA-199a组SD乳鼠心肌细胞的表面积显著小于PE+As-ctl组,P<0.05.结论 miRNA-199a在心肌肥厚过程中可能发挥着调节作用. 目的探討微小RNA(miRNA)-199a在心肌肥厚中的作用.方法 (1)Sprague-Dawley (SD)大鼠12隻,分為腹主動脈縮窄(abdominal aortic constriction,AAC)組(AAC組,n=6)和假手術組(n=6).AAC組通過腹主動脈縮窄術建立大鼠心肌肥厚模型.實時定量聚閤酶鏈反應(qRT-PCR)檢測心肌中部分miRNA錶達的變化.(2)SD乳鼠心肌細胞分為兩組,即miRNA-199a重組腺病毒(Ad-miRNA-199a)組(n=8)和腺病毒載體(Ad-vector)組(n=8),分彆轉染SD乳鼠心肌細胞48 h後qRT-PCR檢測心肌細胞中miRNA-199a以及心肌肥厚標誌分子α肌毬蛋白重鏈(α-myosin heavy chain,αMHC)、β肌毬蛋白重鏈(β-myosin heavy chain,βMHC)、心房鈉尿肽(atrial natriuretic peptide,ANP)編碼基因myh6、myh7、Nppa的錶達變化,併利用免疫熒光分析檢測細胞錶麵積的變化.(3)SD乳鼠心肌細胞分為兩組,即miRNA-199a的反義寡覈苷痠(As-miRNA-199a)組(n=8)和混雜寡覈苷痠(As-ctl)組(n=8),分彆轉染SD乳鼠心肌細胞48 h後qRT-PCR檢測心肌細胞中miRNA-199a的錶達變化.(4)SD乳鼠心肌細胞分為4組,即空白對照組(n=8)、苯腎上腺素組(phenylephrine,PE)(n=8)、PE+As-cd組(n=8)和PE+As-miRNA-199a紺(n=8),分彆轉染SD乳鼠心肌細胞48 h,qRT-PCR檢測心肌肥厚標誌分子編碼基因的錶達變化,免疫熒光檢測細胞錶麵積的變化.結果 (1)qRT-PCR結果顯示,AAC組大鼠造模後1週miRNA-1、miRNA-133、miRNA-181a及miRNA-499的錶達均顯著低于假手術組,而miRNA-199a錶達顯著高于假手術組.(2)qRT-PCR結果顯示,AdmiRNA-199a組SD乳鼠心肌細胞中miRNA-199a的錶達顯著高于Ad-vector組,myh7的錶達亦顯著高于Ad-vector組,而myh6的錶達低于Ad-vector組.免疫熒光顯示Ad-miRNA-199a組SD乳鼠心肌細胞的錶麵積大于Ad-vector組,P<0.05.(3)qRT-PCR結果顯示,As-miRNA-199a組SD乳鼠心肌細胞中miRNA-199a的錶達顯著低于As-ctl組,P<0.05.(4)qRT-PCR結果顯示,PE組Nppa及myh7的錶達量顯著高于空白對照組,而myh6的錶達量低于空白對照組,P<0.05.免疫熒光檢測顯示,PE+As-miRNA-199a組SD乳鼠心肌細胞的錶麵積顯著小于PE+As-ctl組,P<0.05.結論 miRNA-199a在心肌肥厚過程中可能髮揮著調節作用.
목적 탐토미소RNA(miRNA)-199a재심기비후중적작용.방법 (1)Sprague-Dawley (SD)대서12지,분위복주동맥축착(abdominal aortic constriction,AAC)조(AAC조,n=6)화가수술조(n=6).AAC조통과복주동맥축착술건립대서심기비후모형.실시정량취합매련반응(qRT-PCR)검측심기중부분miRNA표체적변화.(2)SD유서심기세포분위량조,즉miRNA-199a중조선병독(Ad-miRNA-199a)조(n=8)화선병독재체(Ad-vector)조(n=8),분별전염SD유서심기세포48 h후qRT-PCR검측심기세포중miRNA-199a이급심기비후표지분자α기구단백중련(α-myosin heavy chain,αMHC)、β기구단백중련(β-myosin heavy chain,βMHC)、심방납뇨태(atrial natriuretic peptide,ANP)편마기인myh6、myh7、Nppa적표체변화,병이용면역형광분석검측세포표면적적변화.(3)SD유서심기세포분위량조,즉miRNA-199a적반의과핵감산(As-miRNA-199a)조(n=8)화혼잡과핵감산(As-ctl)조(n=8),분별전염SD유서심기세포48 h후qRT-PCR검측심기세포중miRNA-199a적표체변화.(4)SD유서심기세포분위4조,즉공백대조조(n=8)、분신상선소조(phenylephrine,PE)(n=8)、PE+As-cd조(n=8)화PE+As-miRNA-199a감(n=8),분별전염SD유서심기세포48 h,qRT-PCR검측심기비후표지분자편마기인적표체변화,면역형광검측세포표면적적변화.결과 (1)qRT-PCR결과현시,AAC조대서조모후1주miRNA-1、miRNA-133、miRNA-181a급miRNA-499적표체균현저저우가수술조,이miRNA-199a표체현저고우가수술조.(2)qRT-PCR결과현시,AdmiRNA-199a조SD유서심기세포중miRNA-199a적표체현저고우Ad-vector조,myh7적표체역현저고우Ad-vector조,이myh6적표체저우Ad-vector조.면역형광현시Ad-miRNA-199a조SD유서심기세포적표면적대우Ad-vector조,P<0.05.(3)qRT-PCR결과현시,As-miRNA-199a조SD유서심기세포중miRNA-199a적표체현저저우As-ctl조,P<0.05.(4)qRT-PCR결과현시,PE조Nppa급myh7적표체량현저고우공백대조조,이myh6적표체량저우공백대조조,P<0.05.면역형광검측현시,PE+As-miRNA-199a조SD유서심기세포적표면적현저소우PE+As-ctl조,P<0.05.결론 miRNA-199a재심기비후과정중가능발휘착조절작용.
Objective To investigate the role of miRNA-199a on cardiac hypertrophy.Methods (1)Male Sprague-Dawley rats were subjected to pressure overload induced by abdominal aortic constriction (AAC,n=6)and quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the change of microRNAs(miRNAs).(2)Neonatal rat ventricular myocytes were isolated from 2-day old Sprague-Dawley rats.The myocytes were divided into two groups:adenovirus miRNA-199a(Ad-miRNA199a) or adenovirus vector(Ad-vector).They were transfected in cardiomyocytes for 48 h using Lipofectamine 2000.qRT-PCR was used to detect the change of myocardial hypertrophy markers α-myosin heavy chain(αMHC,myh6),β-myosin heavy chain(βMHC,myh7)and atrial natriuretic peptide(ANP,Nppa).Software Axio Vision was used to detect the change of cardiomyocytes surface areas.(3)Neonatal rat ventricular myocytes were divided into two groups:antisense oligonucleotide-miRNA-199a(As-miRNA199a) and scramble oligonucleotides (As-ctl). They were transfected to cardiomyocytes respectively for 48 h. qRT-PCR was used to detect the change of miRNA-199a. (4) Neonatal rat ventricular myocytes were divided into four groups : A : control ( ctl), B : phenylephrine ( PE), C : PE + As-ctl, D : PE + As-miRNA199a. qRT-PCR was used to detect the change of myh6 ,myh7 and Nppa. Software Axio Vision was used to detect the change of cardiomyocytes surface areas. Results ( 1 ) qRT-PCR results showed that miRNA-1,miRNA-133, miRNA-181a and miRNA-499 were significantly decreased, while the miRNA-199a was significantly increased at 1 week post AAC hearts compared with the sham group. (2) qRT-PCR results showed that miRNA-199a and myh7 were increased and myh6 was decreased significantly in Ad-miRNA199a group compared with Ad-vector group. The cardiomyocytes surface area was increased in Ad-miRNA199a group detected by immunofluorescence. (3) qRT-PCR results showed that miRNA-199a was significantly decreased in As-miRNA-199a group compared with Ad-vector group. (4) The Nppa and myh7were significantly increased and myh6 was decreased in cardiomyocytes stimulated by PE for 48 h. The cardiomyocytes surface area determined by immunofluorescence was increased in PE + As-miRNA-199a groups compared with PE + As-ctl groups. Conclusion miRNA-199a may play a regulatory role in cardiac hypertrophy.