过表达CUEDC2人脑胶质瘤细胞系U251的构建

发布时间：2018-06-29 21:28

本文选题：胶质瘤 + 胶质母细胞瘤　；参考：《山东医药》2017年27期

【摘要】：目的构建过表达CUE结构域蛋白2(CUEDC2)的人脑胶质瘤U251细胞系。方法 PCR扩增CUEDC2基因全长序列,将其连接于GV358载体质粒。酶切鉴定GV358-CUEDC后采用慢病毒包装三质粒系统(GV358-CUEDC2、Helper 1.0、Helper 2.0)转染293T细胞。转染48、72 h后收集病毒上清并感染U251细胞。将U251细胞分为空白对照组、过表达CUEDC2组、空载体组。空白对照组不进行感染操作,过表达CUEDC2组、空载体组分别感染GV358-CUEDC和GV-vector。流式筛选后采用实时荧光定量PCR法检测U251细胞中的CUEDC2 mRNA,采用Western blotting法检测CUEDC2蛋白。结果 GV358-CUEDC酶切结果显示在900 bp处有一条明显条带,CUEDC2测序结果与Gen Bank数据库比对完全一致。流式筛选感染GV358-CUEDC慢病毒的U251细胞,得到阳性率95%以上的U251细胞。U251细胞在感染GV358-CUEDC2及GV-vector病毒后荧光显微镜下均可见绿色荧光。空白对照组、空载体组、过表达CUEDC2组U251细胞中CUEDC2 mRNA相对表达量分别为5.290±0.663、0.823±0.059、1,CUEDC2蛋白相对表达量分别为0.48±0.020、0.51±0.015、0.92±0.150,过表达CUEDC2组CUEDC2 mRNA及蛋白相对表达量均高于空载体组和空白对照组(P均0.01)。结论成功构建了过表达CUEDC2的人脑胶质瘤U251细胞系。
[Abstract]:Objective to construct a human glioma U251 cell line overexpressing cue domain protein 2 (CUEDC2). Methods the full-length sequence of CUEDC2 gene was amplified by PCR and ligated into GV358 vector plasmid. GV358-CUEDC was digested and transfected into 293T cells by lentivirus packaging triple plasmid system (GV358-CUEDC2Helper 1.0 Helper 2.0). The virus supernatant was collected and infected with U251 cells for 72 h after transfection. U251 cells were divided into blank control group, overexpression CUEDC2 group and empty vector group. The blank control group was infected with GV358-CUEDC and GV-vectorrespectively. After flow screening, CUEDC2 mRNAs in U251 cells were detected by real-time fluorescence quantitative PCR, and CUEDC2 protein was detected by Western blotting assay. Results the results of GV358-CUEDC digestion showed that there was an obvious band of CUEDC2 at 900 BP, which was completely consistent with the comparison of GenBank database. U251 cells infected with GV358-CUEDC lentivirus were screened by flow cytometry, and more than 95% of U251 cells. U251 cells showed green fluorescence under fluorescence microscope after GV358-CUEDC2 and GV-vector infection. The relative expression of CUEDC2 mRNA in U251 cells in blank control group, empty vector group and overexpression CUEDC2 group was 5.290 卤0.663 3 + 0.823 卤0.059 ~ (-1) and 0.48 卤0.020 ~ 0.51 卤0.015 ~ (0.92 卤0.150), respectively. The relative expression of CUEDC2 mRNA and protein in over-expressed CUEDC2 group was higher than that in empty vector group and blank control group (P0.01). Conclusion A human glioma U251 cell line with overexpression of CUEDC2 was successfully constructed.
【作者单位】：徐州医科大学;
【分类号】：R739.41

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