摘要
目的分析Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响,以探讨脑胶质瘤浸润性生长的机理。方法从U87细胞中扩增Ezrin基因CDS区片段,克隆至表达载体pEGFP-C1中,构建Ezrin基因表达质粒pEGFP-C1/Ezrin。将pEGFP-C1/Ezrin、Ezrin基因shRNA质粒shRNA-Ezrin-2和pEGFP-C1以脂质体LipofectimineTM2000介导分别转染U87细胞,Western blot分析转染细胞中Ezrin蛋白的表达;划痕试验检测转染细胞的迁移情况。结果克隆的Ezrin基因与GenBank中登录的Homo sapiens ezrin(EZR),transcript variant 1,mRNA序列同源性达99%;该基因编码的氨基酸序列与ezrin[Homo sapiens](Sequence ID:ref|NP_003370.2|,Length:586)的一致性为99%,在第S66P、K258R、P265L和K577R位发生变异,读码框正确。pEGFP-C1/Ezrin转染的U87细胞中Ezrin蛋白的相对表达量(1.17)高于shRNA-Ezrin-2转染组(0.47)和pEGFP-C1转染组(0.82)。划痕试验显示,shRNA-Ezrin-2转染组有少量细胞迁移至划痕处,pEGFP-C1/Ezrin转染组细胞几乎占满划痕处。结论 Ezrin基因敲低可阻止U87细胞迁移,其过表达可促进细胞迁移,表明U87细胞浸润性生长与Ezrin基因的表达有关。
Objective To study the relationship between Ezrin gene and infiltrative growth of glioma through Ezrin gene knockdown and over-expression.Methods According to Ezrin gene sequence in GenBank,primers were designed using Prime Primer 5.0 software,with which the gene fragment encoding CDS region of Ezrin gene was amplified from U87 cells and cloned into expression vector pEGFP-1.U87 cells were transfected with the constructed recombinant plasmid pEGFP-C1/Ezrin as well as plasmids shRNA-EZrin-2 and pEGFP-C1 in mediation of LipofectimineTM 2000 respectively,then determined for expression of Ezrin protein by Western blot,and for migration by scarification test.Results The homologies of mRNAs of cloned Ezrin gene were 99% to those of homo sapiens ezrin（EZR） and transcript variant 1 reported in GenBank.The homologies of amino acids encoding by the cloned gene was 99% to that of ezrin [Homo sapiens]（Sequence ID： ref-NP_003370.2-,Length：586）,with variations of S66P,K258R,P265L and K577R,while the opening read frame was correct.The relative expression level（1.17） of Ezrin protein in U87 cells transfected with recombinant plasmid pEGFP-C1 / Ezrin was higher those transfected with shRNA-Ezrin-2（0.47） and pEGFP-C1（0.82）.Scarification test showed migration of a small quantity of U87 cells transfected with shRNA-Ezrin-2 to the wound.However,in pEGFP-C1 / Ezrin transfection group,the wound was almost filled with cells.Conclusion Ezrin gene knockdown blocked,while the over-expression promoted the migration of U87 cells,indicating that Ezrin gene involved in the invasive growth of U87 cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第1期56-59,63,共5页
Chinese Journal of Biologicals
