稳定表达EGFRvIII的U87MG细胞株的建立与鉴定

目的:构建稳定表达表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)的U87MG细胞株。方法:采用电转染方法将pcD-NA4/TO-EGFRvⅢ质粒导入U87MG细胞,加入zeocin筛选,进一步将细胞有限稀释并克隆化培养以建立稳定的转染细胞株。应用RT-PCR、Western blot及细胞免疫荧光染色方法鉴定稳定表达EGFRvⅢ基因的细胞株。结果:经RT-PCR鉴定发现V6号和V24号单克隆细胞株EGFRvⅢ基因mRNA水平高表达,进一步Western blot及细胞免疫荧光染色方法鉴定发现V6号细胞株EGFRvⅢ蛋白水平高表达。结论:成功建立了稳定表达EGFRvⅢ的U87MG细胞株,为进一步的抗肿瘤治疗及其机制研究提供基础。

Establishment and Identification of U87MG Cell Line Stably Expressing EGFRvIII

Objective： To establish a human brain glioblastoma cell line U87MG stably expressing the type III mutation of epidermal growth factor receptor（EGFRvIII）.Methods： The pcDNA4/TO-EGFRvIII plasmid was electrotransfected into U87MG cells using amaxa nucleofection technology.The cell clones were screened by zeocin.The expression of EGFRvIII was detected by RT-PCR,Western blot and immunofluorescence staining.Results： By RT-PCR,two lines of U87MG cells（V6 and V24） were identified highly expressing EGFRvIII in mRNA level.Further detected by Western blot and immunofluorescence staining,V6 cell line was confirmed highly expressing EGFRvIII protein.Conclusions： A stable U87MG cell line expressing EGFRvIII was established.This work provides a basis for further anti-tumor study targeting EGFRvIII.