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目的:建立SMAD2稳定干扰的人胚胎干细胞系。方法:利用包装细胞获得重组的逆转录病毒,感染人类胚胎干细胞,为干扰组ShSMAD2、载体组VECTOR和野生型组WT;经荧光筛选获得阳性克隆;Realtime-PCR检测SMAD2 mRNA的表达。结果:带GFP荧光标记的SMAD2特异性shRNA逆转录病毒感染人类胚胎干细胞后,获得稳定干扰SMAD2表达的人胚胎干细胞系。经检测shSMAD2组SMAD2 mRNA表达较VECTOR和WT组明显降低,VECTOR和WT组之间无明显差异。结论:通过SMAD2特异性shRNA逆转录病毒载体构建了稳定干扰SMAD2的人类胚胎干细胞。
Objective: To establish SMAD2 stable interfering human embryonic stem cell line. Methods: Recombinant retrovirus was obtained from packaging cells and infected with human embryonic stem cells. The interference cells were ShSMAD2, VECTOR and WT. The positive clones were obtained by fluorescence screening. The expression of SMAD2 mRNA was detected by Realtime-PCR. RESULTS: Human embryonic stem cell lines stably interfering with SMAD2 expression were obtained after infection of human embryonic stem cells with GFP fluorescently labeled SMAD2-specific shRNA retrovirus. The expression of SMAD2 mRNA in shSMAD2 group was significantly lower than that in VECTOR and WT groups, but there was no significant difference between VECTOR and WT groups. Conclusion: SMAD2-specific human embryonic stem cells were stably transfected by SMAD2-specific shRNA retroviral vector.