山东医药2017，Vol.57Issue(27)：1-4,4.DOI:10.3969/j.issn.1002-266X.2017.27.001
过表达CUEDC2人脑胶质瘤细胞系U251的构建
Establishment of human glioma U251 cell line with overexpression of CUEDC2
摘要
Abstract
Objective To construct human glioma U251 cell line with overexpression of CUE domain containing 2 (CUEDC2).Methods Full-length CUEDC2 gene fragment was amplified by RT-PCR and linked to GV358 plasmid vector.After identifying GV358-CUEDC,we transfected and packaged it using lentivral packaging system (GV358-CUEDC2,Helper 1.0,and Helper 2.0) into renal epithelial cells (293T) by lipofectamin 2000 in order to obtain the LV-CUEDC2 lentivirus.After 48 and 72 h,we collected the virus supernatant and infected the U251 cells.Cells were divided into the blank control group (without any infection),LV-CUEDC2 group (transfected with GV358-CUEDC),and LV-vector group (transfected with GV-vector).After Flow cytometry sorting,Western blotting and real-time fluorescent quantitative PCR were used to detect the mRNA and protein expression levels of CUEDC2.Results GV358-CUEDC digestion showed that there was an obvious band at 900 bp.CUEDC2 sequencing results were consistent with the GenBank database.The results of flow cytometry confirmed the positive rate up to 95%.LV-CUEDC2 group and LV-vector group cells showed green fluorescence under fluorescence microscope after infection with GV358-CUEDC2 and GV-vector.The CUEDC2 mRNA levels of the blank control group,LV-vector group and LV-CUEDC2group were 5.290±0.663,0.823±0.059,and 1,respectively.The CUEDC2 protein expression levels of the three groups were 0.48±0.02,0.51±0.015,and 0.92±0.15,respectively.The CUEDC2 protein and mRNA expression levels of LV-CUEDC2 group were both higher than those of the other groups (both P<0.01).Conclusions We successfully constructed the human glioma U251 cell line with CUEDC2 overexpression.关键词
胶质瘤/胶质母细胞瘤/CUE结构域蛋白2/慢病毒Key words
glioma/glioblastoma/CUE domain containing 2/lentivirus分类
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唐传喜,刘鑫峰,李风,高殿帅..过表达CUEDC2人脑胶质瘤细胞系U251的构建[J].山东医药,2017,57(27):1-4,4.
