构建干扰多梳状蛋白2的人胶质瘤U251细胞系  

Construction of Human Glioma U251 Cell Line that Interferes with Polycomb Like 2

作　　者：王菲 高永英[2] 武艳玮 徐丽珲 马杰 刘焕 曹相玫 WANG Fei;GAO Yongying;WU Yanwei;XU Lihui;MA Jie;LIU Huan;CAO Xiangmei(Department of Pathology,School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;Department of Neurology,Ningxia People’s Hospital,Yinchuan 750004,China;Institute of Basic Medicine Science,Xi’an Medical University,Xi’an 710021,China)

机构地区：[1]宁夏医科大学基础医学院病理学系,银川750004 [2]宁夏回族自治区人民医院神经内科,银川750002 [3]西安医学院基础医学部,西安710021

出　　处：《宁夏医科大学学报》2019年第6期556-560,共5页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81460433);宁夏自然科学基金(NZ15177);宁夏高等学校一流学科建设(宁夏医科大学西部一流建设学科基础医学)资助项目(NXYLXK2017B07);陕西省自然科学基础研究计划-青年人才项目(2016JQ8001)

摘　　要：目的建立稳定干扰多梳状蛋白2(polycomb like 2,PCL2)的人胶质瘤U251细胞系。方法针对PCL2基因的shRNA克隆至miR30前体骨架shRNA表达载体,用PCR鉴定载体构建成功后转入HEK293细胞,得到重组腺病毒Ad5-E1-AmU6-PCL2 shRNA-E3-CMV-eGFP;将重组腺病毒转染至U251细胞中,分组为空白对照组、空载体组和PCL2 shRNA组,滴度梯度设置为MOI(20∶1,50∶1,100∶1),使用荧光显微镜观察腺病毒感染效率;用Western blot检测U251细胞内shRNA PCL2表达载体的基因沉默效率。结果使用小RNA干扰技术,成功构建了表达PCL2 shRNA的重组腺病毒;荧光显微镜观察发现MOI(50∶1)时,U251细胞感染效率最高,细胞生长状态良好。Western blot结果显示PCL2 shRNA重组腺病毒组的PCL2蛋白水平较空白对照组和空载体组明显降低,以100∶1时的PCL2表达量最低,干扰效果最显著。结论成功构建的PCL2 shRNA重组腺病毒能够在体外有效感染U251细胞,降低U251细胞中PCL2的蛋白表达并筛选出稳定沉默的细胞系。Objective To establish a human glioma U251 cell line stably interfering with polycomb like 2(PCL2).Methods The shRNA of PCL2 gene was cloned into the shRNA expression vector of mir30 precursor skeleton.The PCR identification vector was successfully constructed and then transferred into HEK293 cells for preparation and purification.The recombinant adenovirus Ad5-E1-AmU6-PCL2 shRNA-E3-CMV-eGFP was obtained.The recombinant adenovirus was transfected into U251 cells and divided into blank control group,empty vector group and PCL2 shRNA group.The titer gradient was set to MOI(20∶1;50∶1;100∶1).The efficiency of adenovirus infection was observed by fluorescence microscope.Western blot was used to detect the gene silencing efficiency of shRNA PCL2 expression vector in U251 cells.Results The recombinant adenovirus expressing PCL2 shRNA was successfully constructed by using small RNA interference technique.Fluorescence microscopy showed that U251 cells had the highest infection efficiency and good growth state when MOI(50∶1)was observed.Western blot showed that the level of PCL2 protein in PCL2 shRNA recombinant adenoviral group was significantly lower than that in blank control group and empty vector group,and the expression of PCL2 was the lowest at 100∶1,and the interference effect was the most significant.Conclusion The successfully constructed PCL2 shRNA recombinant adenovirus can effectively infect U251 cells in vitro,reduce the expression of PCL2 protein in U251 cells and screen stable silent cell lines.

关 键 词：多梳状蛋白2 U251细胞 SHRNA microRNA-30 胶质瘤 

分 类 号：R739.41[医药卫生—肿瘤]