慢病毒介导shRNA沉默E2-2基因促进内皮前体细胞增殖  

Effect of lentivirus-mediated E2-2 gene silencing on proliferation in endothelial progenitor cells

作　　者：刘晓丽[1] 马阳[1] 梁源[1] 喻杨[2] 梁云华[1] 王红[1] 

机构地区：[1]成都军区昆明总医院干部病房,昆明650032 [2]第三军医大学新桥医院心血管内科,全军心血管病研究所,重庆400037

出　　处：《第三军医大学学报》2015年第22期2261-2266,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81270224);第三军医大学青年基金(2012D288)~~

摘　　要：目的构建小鼠转录因子基因E2-2 RNA干扰（RNA interference,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）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18-20 g。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,48 h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,E2-2 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。Objective To construct RNA interference（ RNAi） lentiviral vector targeting murine transcription factor E2-2 gene and investigate its effect on the growth and proliferation of endothelial progenitor cells（ EPCs）. Methods Murine EPCs were isolated from bone marrow,cultured and identified. Three potential RNAi sites were screened based on the mRNA sequence of E2-2 gene and synthesized. The 3synthesized siRNAs were transferred into the EPCs, and reverse transcription-polymerase chain reaction（ RT-PCR） was used to analyze the inhibitory effect of the target gene so as to identify the optimal siRNA sequence. Then the short-hairpin RNA（ shRNA） oligonucleotide targeting the optimal siRNA sequence was designed,synthesized and inserted into FT203-PLVX plasmid to construct FT203-PLVX / E2-2shRNA lentiviral vector. After identifying by sequencing,FT203-PLVX / E2-2shRNA plasmid was transferred into 293 cells to produce lentiviral particles. The EPCs were transfected with the lentiviral particles,and the expression of green fluorescent protein（ GFP） in the transfected cells was observed with an inverted microscope. Cell count kit-8（ CCK-8） was used to test the growth and proliferation of the transfected cells. The expression of E2-2,proliferating cell nuclear antigen（ PCNA） gene and its encoding protein in the transfected cells were quantitatively detected by RT-PCR and Western blotting. Results Murine EPCs were isolated from the bone marrow,cultured and identified. The optimal siRNA sequence CGTCAGCTAGTGTTTCTAA was confirmed.Sequencing results showed that lentiviral vector FT203-PLVX / E2-2shRNA was constructed successfully. The transfected cells significantly expressed GFP. The CCK-8 results revealed that the growth and proliferation of EPCs were faster after E2-2 gene silencing,especially from 48 h after transfection（ P 0. 01）. The results of RT-PCR,Western blotting and quantitative analysis showed that the lentiviral vector FT203-PLVX / E2-2shRNA effectively silenced E2-2 gene and up-regulat

关 键 词：E2-2基因 RNAI 内皮前体细胞 慢病毒 

分 类 号：R322.12[医药卫生—人体解剖和组织胚胎学]