| 摘要: |
| [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。 |
| 关键词: 心肌细胞 腺病毒 microRNAs miR-1 |
| DOI：10.3724/SP.J.1008.2010.01161 |
| 投稿时间：2010-06-01修订日期：2010-10-06 |
| 基金项目:国家自然科学基金(30828006). |
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| Role of miR-1 in regulating cardiomyocyte growth |
| XU Xu-dong, SONG Xiao-wei,QIN Yong-wen* |
| (Department of Cardiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China) |
| Abstract: |
| [Abstract] 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). Methods 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. Methods 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. |
| Key words: cardiomyocyte adenovirus microRNAs miR-1 |
