【摘要】 目的构建再生基因蛋白3β(regenerating islet-derived protein 3 beta,Reg3β)干扰载体,并在H9C2大鼠心肌细胞中优化转染条件,鉴定抑制效果,为深入探索Reg3β在心血管疾病的作用提供有利的工具。方法设计合成Reg3β干扰序列,在5’端加入一个SacⅠ的酶切位点,3’端加入LOOP环序列和反向互补序列,构建pG1.2-Reg3βshRNA沉默载体。接着分别用Lipofectamine2000和Lipofectamine3000优化摸索转染大鼠H9C2心肌细胞的效率,以q-PCR和Western blot法检测H9C2细胞内Reg3β的表达量。结果设计合成了3对Reg3β干扰序列,从中筛选出抑制效率最高达67.93%的序列,酶切和测序结果证实成功构建了pG1.2-Reg3βshRNA。经过优化比较发现,pG1.2-Reg3βshRNA沉默载体Lipofectamine3000比例为1μg∶3μl时,其转染效率最高(50%以上)。q-PCR和Western blot法检测结果显示,最佳干扰载体在最高转染条件下可使H9C2细胞Reg3β表达量降低50%以上。结论成功构建了pG1.2-Reg3βshRNA沉默载体,并在H9C2细胞中有效干扰Reg3β的表达,可用于探讨Reg3β在心血管疾病中的作用。更多还原

【Abstract】 Objective To build and analyze the regenerating islet-derived protein 3 beta( Reg3β) shRNA in rat H9C2 cells for studying the function of Reg3β gene in cardiovascular disease. Methods The shRNAs target fragments of the rat Reg3β gene were designed and synthesized. To construct the pG1. 2-Reg3β shRNA,the restriction site of Sac I was added at the 5’-end,while a LOOP sequence and a reverse complement sequence were added at the 3’-end. The pG1. 2-Reg3β shRNA was transfected to H9C2 cells from Lipofectamine 2000 and Lipofectamine3000. q-PCR and Western Blot were used to detect the expression of Reg3β in H9C2 cells.Results From three pairs of Reg3β shRNA,a vector with the highest interfering efficacy( 67. 93%) was a successful pG1. 2-Reg3βshRNA. The relatively high transfection efficiency was found in the group with the ratio of shRNA/Lipofectamine3000 = 1μg∶ 3μl. The relatively protein expression of Reg3β was significantly decreased in H9C2 cells by pG1. 2-Reg3β shRNA. Conclusion pG1. 2-Reg3β shRNA was successfully builder and expressed in H9C2 cells,which provide a useful tool for investigating the potential protective role of Reg3β in cardiovascular disease.更多还原