用小双链RNA抑制转化入HUVEC中绿色荧光蛋白基因的表达

siRNA suppresses the green fluorescent protein (GFP) gene expression in human umbilical vein endothelial cells

目的:建立稳定表达绿色荧光蛋白(GFP)的人脐静脉血管内皮细胞(HUVECs),研究小干扰RNA(siR-NA)对HUVECs中GFP表达的抑制作用。方法:用lipofectamine2000将编码GFP的质粒pN3-EGFP转入HUVECs中。用G418筛选并维持已转化pN3-EGFP的HUVEC(HUVEC-GFP)。利用T7RNA转录试剂盒,制备可抑制GFP基因表达的siRNA(GFPsiRNA)和无关对照的RNA(control siRNA)。用oligofectamine将siRNA转入HUVEC-GFP中。继续培养48h后,检测HUVEC-GFP中GFP蛋白和mRNA表达水平。结果:用G418筛选获得了HUVEC-GFP细胞株,可以观察到GFP的稳定表达。HUVEC-GFP转化siRNA后48h,GFP的荧光强度明显下降,而对照组的荧光强度无明显下降。半定量RT-PCR检测显示,GFPsiRNA对GFP mRNA表达有较强的抑制作用,抑制率达40%,而control siRNA对GFP mRNA表达水平无明显的抑制作用。结论:利用体外转录合成的siRNA能有效地抑制HUVECs中GFP的表达。

AIM： To establish human umbilical vein endothelial cells （HUVECs） to express green fluorescent protein （GFP）, and to study the suppression of GFP by siRNA in HUVECs. METHODS： Using lipofectamine 2000 to transform plasmid pN3 - EGFP encoding GFP into HUVECs. The HUVEC containing pN3 - EGFP, named HUVEC - GFP, was screened and selected by antibiotic G418. Using in vitro transcription T7 kit, GFPsiRNA targeting GFP mRNA and control- siRNA used as control were synthesized. The siRNAs were transfected into HUVEC - GFP with oligofectmnine. 48 h later, the expression levels of GFP protein and mRNA in HUVEC- GFP were determined. RESULTS： The HUVEC- GFP was screened to express GFP in the presence of G418. The agarose gel electrophoresis analysis showed that the siRNAs prepared were integrated. 48 h after transfection with siRNAs, compared to control group, the level of GFP fluorescence was obviously decreased in the HUVEC - GFP transfected with GFP- siRNA. The results of RT - PCR detection showed that GFP mRNA expression was obviously suppressed by GFPsiRNA at the rate of 40%, and no obvious suppression of GFP mRNA expression was found in the HUVEC - GFP transfected with control siRNA. CON- CLUSION： The siRNA targeting GFP mRNA, synthesized in vitro, efficiently suppresses the GFP expression in HUVECs.