miR-22心肌特异转基因斑马鱼的构建及心肌肥厚的评估

Construction of Cardiac-specific miR-22 Transgenic Zebrafish Line and Evaluation of Cardiac Hypertrophy

目的:构建miR-22心肌特异转基因斑马鱼系,在体评估miR-22对于心肌肥厚的作用。方法:构建pTol2-CMLC2-miR-22-IRES-EGFP表达载体。通过显微注射的方法将tol2重组质粒于一细胞期注射入斑马鱼受精卵胚胎中,荧光筛选获得心肌特异表达绿色荧光的斑马鱼胚胎,并稳定表达传代。然后对稳定传代的成年斑马鱼心脏进行心肌肥厚及心功能的检测。结果:成功建立了miR-22心肌特异转基因斑马鱼系,通过定量PCR确定心肌中miR-22表达升高,荧光显微镜观察发现斑马鱼心肌出现绿色荧光。miR-22心脏特异过表达的转基因鱼系的成年鱼与野生对照组相比,出现了心肌肥厚的现象,心肌肥厚分子标志物nppa、myh7明显升高。斑马鱼心脏病理切片结果同样显示出miR-22心肌特异转基因斑马鱼出现了心肌肥厚的现象。结论:成功构建了miR-22心肌特异转基因斑马鱼,为研究心肌中miR-22的生物学功能提供了重要的工具,并证明miR-22心脏特异过表达会引起斑马鱼心肌肥厚。

Objective： Construction of cardiac-specific miR-22 transgenic zebrafish line and evaluation of cardiac hypertrophy. Methods： To construct the pTo12-CMLC2-miR-22-IRES-EGFP expression vector, The To12 recombinant plasmid was injected into zebrafish embryos in one-cell stage by microinjection, and the embryos expressing cardiac-specific green fluorescence were screened. Different generation were selected and raised to get a stable transgenic line. And then heart section and analysis of heart function were performed on the stably miR-22 overexpressed adult zebrafish. Results： The cardiac-specific miR-22 transgenic zebrafish line was constructed successfully, in which over-expression of miR-22 in cardiac tissues was identified via quantitative PCR and displayed the phenomenon of cardiac hypertrophy. Fluorescence microscopy revealed a green fluorescent zebrafish heart. Molecular markers of cardiac hypertrophy, nppa and myh7, were significantly increased, miR-22 cardiac-specific transgenic zebrafish cardiac pathology results also appeared hypertrophy phenomenon. Conclusions： We successfully constructed a cardiac-specific miR-22 transgenic zebrafish to study the function of miR-22 in myocardial tissues and identified miR-22 cardiac-specific overexpression would cause cardiac hypertrophy.