miR-1在心肌细胞生长中的调节作用  

Role of miR-1 in regulating cardiomyocyte growth

作　　者：许旭东[1] 宋晓伟[1] 秦永文[1] 

机构地区：[1]第二军医大学长海医院心血管内科,上海200433

出　　处：《第二军医大学学报》2010年第11期1161-1164,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30828006)~~

摘　　要：目的构建并鉴定microRNA-1(miR-1)的腺病毒表达载体,并探讨其在心肌肥厚中的介导作用。方法 PCR扩增含大鼠miR-1前体的DNA片段,并将其克隆到腺病毒穿梭质粒pAdTrack。pAdTrack经PmeⅠ酶切线性化后与腺病毒骨架质粒pAdEasy-1在BJ5183菌中进行同源重组。重组质粒pAd-precusor-miR-1线性化后,转染293A细胞,进行病毒包装,得到重组腺病毒颗粒Ad-miR-1。Ad-miR-1与Ad-GFP病毒转染培养的乳鼠心肌细胞,通过实时荧光定量PCR方法检测miR-1的表达效率,并分析心肌细胞表面积及肥厚标志物ANP(Nppa)、β-MHC(myh7)的基因表达变化。结果基因测序及酶切鉴定证实重组Ad-precursor-miR-1腺病毒载体构建成功,腺病毒Ad-miR-1转染心肌细胞后,实时荧光定量PCR方法证实腺病毒Ad-miR-1能够显著提高心肌细胞内miR-1的表达水平,减小心肌细胞表面积及Nppa、myh7的表达。结论利用同源重组方法构建的miR-1腺病毒能够提高心肌细胞内miR-1的表达,抑制心肌细胞生长。Objective To construct and identify miR-1 adenovirus vector,and to analyze its effect on cardiac hypertrophy.Methods The primers of miR-1 precursor were designed for PCR amplification,and the PCR products were cloned into adenovirus shuttle plasmid pAdTrack and linearized by enzyme PmeⅠ;the resultant plasmid was co-transfected into E.coli BJ5183 cells with adenovirus backbone plasmid pAdEasy-1 for homologous recombination.Then the recombinant plasmid was identified,linearized and packaged into QBI-293A cells to amplify the recombinant adenovirus Ad-miR-1,which was then used to infect cardiomyocytes.Real-time quantitative PCR was used to observe the expression of miR-1 and two hypertrophic markers,the atrial natriuretic peptide（Nppa） and β-myosin heavy chain（myh7）,in cultured primary cardiomyocytes.Cell surface area was analyzed using software AxioVision 4.7.1（Carl Zeiss）.Results Sequencing and enzyme digestion showed that the miR-1 recombinant plasmid was successfully constructed.Real-time quantitative PCR confirmed that adenovirus Ad-miR-1 significantly enhanced intracellular miR-1 expression in cardiomyocytes and reduced cell surface area and the expression of Nppa and myh7.Conclusion The adenovirus expressing miR-1 has been successfully constructed and it can be transfected into cardiomyocytes to increase the expression of miR-1,thus inhibiting cardiomyocyte growth.

关 键 词：心肌细胞 腺病毒 MICRORNAS MIR-1 

分 类 号：R542.2[医药卫生—心血管疾病]