慢病毒介导shRNA沉默E2-2基因对内皮前体细胞生长与增殖的影响.docVIP

慢病毒介导shRNA沉默E2-2基因对内皮细胞生长与增殖的影响 都军区昆明总医院干部病房650032) [摘要] 目的 构建 RNAi慢病毒载体,并观测其沉默E2-2基因对内皮前体细胞（endothelial progenitor cells, EPCs）生长与增殖的影响。方法 首先分离、培养小鼠骨髓EPCs，并予以鉴定。针对E2-2基因mRNA序列，筛选3个潜在的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E2-2shRNA慢病毒。将该重组慢病毒感染EPCs，倒置显微镜。EPCs得以分离、培养并予以鉴定。筛选到E2-2基因的最佳干扰靶序列为CGTCAGCTAGTGTTTCTAA；测序证实，成功构建FT203-PLVX/E2-2 shRNA慢病毒载体。荧光观察显示，被感染细胞显著表达GFP。CCK-8检测表明，与对照细胞比较，沉默E2-2的EPCs的生长与增殖加快，48 h开始变得更为明显（P0.01）；mRNA和蛋白水平检测及定量分析证实，E2-2 shRNA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、培养并予以鉴定。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的生长与增殖并上调其核抗原PCNA的表达。 [关键词] E2-2基因；RNA干扰The effect of E2-2 gene silencing by lentvirus-mediated shRNA on the growth and proliferation of endothelial progenitor cells LIU Xiao-li, MA Yang, GAO Yuan,YANG Hai-jie, LIANG Yun-hua, Wang Hong (Cadres’Wards, Kunming General Hospital of chengdu Military Command，Kunming,Yunnan province, 650032, China) [Abstract] Objective To construct a RNA interference (RNAi) lentiviral vector targeting murine transcription factor E2-2 gene and investigate its effect on growth and proliferation of endothelial progenitor cells (EPCs). Methods Murine EPCs of bone marrow were isolated, cultured and identified. Three potential RNAi sites were screened based on the mRNA sequence of E2-2 gene and synthesized. To identify the optimal siRNA sequence by reverse transcription polymerase chain reaction (RT-PCR), these three synthesized siRNA were transfected into EPCs. Then the short hair RNA (shRNA) oligonucleotide targeting the optimal siRNA sequence was designed, synthesized and inserted into FT203-PLVX plasmid to form FT203-PLVX/E2-2shRNA lentiviral vector. After identified by sequencing, FT203-PLVX/E2-2shRNA plasmid was transfected into 293 cells to produce lentiviral particles. The lentiviral particles were transfected into EPCs and the expression of green fluorescen