文章摘要
魏飞宇,吕 丽,马方方,周 蕾.Popdc2表达下调通过Akt磷酸化促进新生大鼠心肌细胞增殖[J].南京医科大学学报,2015,(7):938~944
Popdc2表达下调通过Akt磷酸化促进新生大鼠心肌细胞增殖
Proliferation of neonatal cardiomyocytes by knockdown of Popdc2 via Akt-phosphorylation
投稿时间:2015-01-29  
DOI:10.7655/NYDXBNS20150708
中文关键词: Popdc2  心肌细胞  增殖  Akt  转录因子
英文关键词: Popdc2  cardiomyocytes  proliferation  Akt  transcription factor
基金项目:国家自然科学基金(81370280)
作者单位
魏飞宇 南京医科大学第一附属医院心脏科,江苏 南京 210029 
吕 丽 昆明医科大学第三附属医院,云南省肿瘤医院生物治疗中心,云南 昆明 650118 
马方方 南京医科大学第一附属医院心脏科,江苏 南京 210029 
周 蕾 南京医科大学第一附属医院心脏科,江苏 南京 210029 
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中文摘要:
      目的:探讨Popdc2对新生大鼠心肌细胞增殖作用及其机制?方法:分离出生后第0?7?14天大鼠心脏组织,荧光定量逆转录-聚合酶链反应(qRT-PCR)检测心脏组织中Popdc2 mRNA表达情况?分离出生后0~2 d SD大鼠原代心肌细胞和成纤维细胞,qRT-PCR检测Popdc家族mRNA在心肌细胞表达情况?Popdc2 mRNA在心肌细胞和成纤维细胞表达情况;大鼠原代心肌细胞分别转染阴性对照小干扰RNA(NC组)和Popdc2特异性小干扰RNA(si-Popdc2组),EdU实验?Ki67染色检测心肌细胞增殖,免疫荧光检测心肌细胞特异性蛋白辅肌动蛋白(actinin)和心肌钙蛋白2(TNNT2)表达,qRT-PCR检测转染小干扰RNA后心肌细胞Popdc2及转录因子TBX20?TBX5 mRNA表达?增殖抗原蛋白Ki67 mRNA表达情况,Western blot检测总Akt及磷酸化Akt蛋白表达水平?结果:① Popdc家族中Popdc2 mRNA在心肌细胞表达最高(P均<0.01),且随着出生后天数增加Popdc2表达逐渐升高(P均<0.01),Popdc2 mRNA在心肌细胞表达高于成纤维细胞(P < 0.05);②与对照组相比,沉默Popdc2可促进原代心肌细胞DNA合成(P < 0.05),并增加Ki67阳性心肌细胞数量(P < 0.05);③qRT-PCR显示沉默Popdc2可上调转录因子TBX20(P < 0.01)?TBX5(P < 0.05)的mRNA表达,沉默Popdc2促进Ki67的mRNA表达 (P < 0.05),Western blot结果显示沉默Popdc2可促进Akt蛋白磷酸化,Akt308(P < 0.05),Akt473(P < 0.001)?结论:Popdc2表达下调可能通过调节转录因子表达及Akt蛋白磷酸化促进新生大鼠心肌细胞增殖,Popdc2 有可能成为心肌损伤后修复和再生治疗靶点?
英文摘要:
      Objective:To explore whether and how the knockdown of Popdc2 with siRNA produced changes in the proliferative activity of neonatal rat cardiomyocytes. Methods:Postnatal of 0,7,14 day of Sprague Dawley rats cardiac tissue was prepared,qRT-PCR was performed to detect the Popdc2 relative mRNA expression level. Neonatal rat cardiomyocytes and fibroblast were prepared from the ventricles of Sprague Dawley rats aged 0~2 days,qRT-PCR was performed to detected Popdc family member mRNA expression level in cardiomyocytes and Popdc2 mRNA expression level in cardiomyocytes and fibroblast;negative control(NC)and Popdc2 small interference RNAs (si-Popdc2) were transferred into neonatal rat cardiomyocytes,EdU incorporation assay and Ki67 staining were used to detect cell proliferation,qRT-PCR was performed to detect the Popdc2,TBX20,TBX5,and Ki67 relative mRNA expression level,Western blot was used to test the total Akt and phosphorylation Akt protein expression level. Results:This study showed that Popdc2 mRNA expression level was the highest of Popdc family member in cardiomyocytes (P < 0.01). The expression was continuely increased after postnatal(P < 0.01),Popdc2 mRNA expression level was higher in cardiomyocytes compared with fibroblast(P < 0.05);EdU incorporation assay showed that knockdown of Popdc2 promoted cardiomyocytes DNA systhesis(P < 0.05),immune staining showed knockdown of Popdc2 increased the Ki67 position cardiomyocytes number (P < 0.05),qRT-PCR showed that knockdown of Popdc2 upregulated transcription factor TBX20(P < 0.01),TBX5(P < 0.05)),and proliferation protein Ki67(P < 0.05)mRNA expression level,Western blot showed that knockdown of Popdc2 significantly increased phosphorylation of Akt protein level,Akt308 (P < 0.05),Akt473 (P < 0.001). Conclusion:The present study demonstrated that knockdown of Popdc2 produced a significant increase in the proliferation of neonatal cardiomyocytes,and may via Akt phosphorylation and regulate cardiac transcription factor,suggesting that Popdc2 may become a therapeutic target for cardiac repair and heart regeneration.
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