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IL-17可以通过多种途径促进肿瘤发展,前期工作发现胶质瘤组织中IL-17高表达,本研究拟构建IL-17真核表达载体pEGFP-N1-IL-17,并且稳定转染胶质瘤细胞株U87MG,为研究IL-17在胶质瘤中作用提供基础。取血小板减少性紫癜自愿患者外周血2ml,Ficoll分离PBMC,提取RNA后经含BamHⅠ及SalⅠ酶切位点引物逆转录成cDNA,连接T载体,酶切后与经相同酶切的载体pEGFP-N1连接,卡那霉素筛选,重组载体经Xfect试剂转染胶质瘤细胞U87MG,以G418筛选,单克隆阳性株扩大培养,经荧光、Real Time PCR及ELISA鉴定。构建真核表达载体pEGFP-N1-IL-17测序正确。经荧光、Real Time PCR、ELISA检测稳定转染细胞株pEGFP-N1-IL-17-U87MG成功,IL-17转染后U87MG细胞MCP-1分子表达下调。因此,本研究成功构建IL-17真核表达载体pEGFP-N1-IL-17,并稳定转染U87MG,为研究IL-17在胶质瘤中作用提供了基础。
IL-17 can promote tumor development in a variety of ways. Previous work found that IL-17 was highly expressed in glioma tissue. In this study, IL-17 eukaryotic expression vector pEGFP-N1-IL-17 was constructed, The tumor cell line U87MG, to provide a basis for studying the role of IL-17 in glioma. Peripheral blood of patients with thrombocytopenic purpura was taken 2ml, PBMCs were isolated by Ficoll, RNA was extracted and then reverse transcribed into cDNA by BamHⅠ and SalⅠ restriction sites, ligated with T vector, digested with the same vector pEGFP-N1 The recombinant vector was transfected into U87MG glioma cells by Xfect reagent, and then screened by G418. The monoclonal positive strain was expanded and identified by fluorescence, Real Time PCR and ELISA. The eukaryotic expression vector pEGFP-N1-IL-17 was constructed and sequenced correctly. The expression of MCP-1 in U87MG cells was down-regulated after IL-17 transfection by fluorescence, Real Time PCR and ELISA. Therefore, we successfully constructed IL-17 eukaryotic expression vector pEGFP-N1-IL-17 and stably transfected it into U87MG, which provided a basis for studying the role of IL-17 in glioma.