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慢病毒介导shRNA沉默E2-2基因促进内皮前体细胞增殖

, PP. 2261-2266

Keywords: E-基因,RNAi,内皮前体细胞,慢病毒

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Abstract:

目的构建小鼠转录因子基因E2-2RNA干扰(RNAinterference,RNAi)慢病毒载体,并观测其沉默E2-2基因对内皮前体细胞(endothelialprogenitorcells,EPCs)增殖的影响。方法SPF级昆明小鼠20只,雌雄不拘,4周龄,体质量18~20g。分离、培养小鼠骨髓EPCs,并予以鉴定。针对E2-2基因mRNA序列,筛选3个siRNA干扰靶点并予以合成;将合成的siRNA导入EPCs,用RT-PCR法检测其对靶基因的抑制效果,以确定最佳siRNA。设计并合成针对最佳siRNA靶序列的shRNA,连入FT203-PLVX载体,构建慢病毒载体FT203-PLVX/E2-2shRNA,并予以测序鉴定。测序正确者经293细胞包装,形成具高效感染力的FT203-PLVX/E2-2shRNA慢病毒。将该重组慢病毒感染EPCs,倒置显微镜观测被感染细胞的绿色荧光蛋白(GFP)的表达变化、CCK-8法检测被感染细胞的增殖情况;在mRNA和蛋白水平分别检测并定量分析被感染细胞的E2-2及核抗原PCNA基因及其编码蛋白的表达变化。结果小鼠骨髓EPCs得以分离、培养与鉴定。筛检到E2-2基因的最佳干扰靶序列为CGTCAGCTA-GTGTTTCTAA;测序证实,成功构建FT203-PLVX/E2-2shRNA慢病毒载体。荧光观察显示,被慢病毒感染的EPCs明显表达GFP。CCK-8检测表明,与对照细胞比较,沉默E2-2的EPCs的增殖加快,48h开始变得更为明显(P<0.01);mRNA和蛋白水平检测及定量分析结果显示,E2-2shRNA慢病毒能有效沉默EPCs的E2-2,并上调其核抗原PCNA基因及其编码蛋白的表达。结论小鼠骨髓EPCs得以分离、培养与鉴定;成功构建E2-2基因RNAi慢病毒载体,该载体能有效沉默EPCs的E2-2基因,促使EPCs的增殖并上调其核抗原PCNA的表达。

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