摘要
目的 探讨靶向表皮生长因子受体(epidermal growth factor receptor,EGFR)的RNA干涉(RNAi)技术抑制恶性胶质瘤体外细胞系U25l的EGFR表达后,在体内外对U251细胞生长抑制作用。方法 在人EGFR开放阅读框的细胞内区酪氨酸激酶结构域选择1个siRNA靶序列、进行短发夹RNA(shRNA)表达载体psiRNA-2400的构建,并以含有随机序列的psiRNA-scr表达质粒为对照进行了脂质体介导的U251人脑恶性胶质瘤细胞系表达。应用RT-PCR检测EGFR表达水平,应用MTT法、流式细胞术和Matrigel基质生长实验评价肿瘤细胞转染前后的生物学行为。进一步应用裸鼠皮下荷瘤模型观察脂质体介导shRNA基因治疗对U251细胞生长抑制作用。对肿瘤组织应用免疫组化的方法进行EGFR、PCNA和GFAP表达比较。结果脂质体介导、靶向EGFR的shRNA可显著抑制U251细胞ECFR表达,Mr兀’法分析显示psiRNA-2400转染组细胞生长抑制率为68%;与对照组和psiRNA-scr转染组比较,细胞周期分析结果表明psiRNA-2400转染组G0~G1期细胞数没有明显变化,进入s期的细胞数较对照组减少了12.7%~13%,而进入G2期细胞则增加了10.5%~11、7%。Matrigel基质生长实验显示对照组和psiRNA-scr转染组细胞呈正常形态贴壁生长,而psiRNA-2400转染组细胞不能贴壁生长,呈团块状簇集生长。裸鼠皮下荷瘤模型实验显示psiRNA-2400显著抑制皮下肿瘤生长,组织病理学分析显示治疗组EGFR表达下降、PCNA标记指数降低而GFAP表达上调。结论靶向EGFR的RNAi技术可以显著抑制U251细胞的EGFR表达,在体内外对U251细胞生长均产生明显抑制作用。因此,shRNA表达质粒介导的基因治疗可以成为胶质瘤靶向性EGFR治疗的新策略。
Objective To investigate growth inhibition effect on tumor growth of U251 glioma cell with EGFR overexpression by small hairpin RNA targeting EGFR. Methods One short hairpin RNA (shRNA) expression constructs, psiRNA-2400, which targeted sequences of human EGFR introcellular catalytic domain (2400-2420), were transfected into human malignant glioma U251 cells as mediated by Lipofectamine. The psiRNA-scr which contained scrambled sequences were set as negative control. Reverse transcript polymerase chain reaction (RT-PCR) was rendered to detect EGFR expression. The proliferative activities of the tumor cells were measured by MTT, flow cytometry and in vitro growth on matrigel matrix. The tumor growth inhibition effect was further studied in vivo. PsiRNA-scr and psiRNA-2400 were injected intratumorally into established subcutaneous U251 glioma in nude mice mediated by lipofectamine, respectively. During observation period of 30 days, tumor volume was measured every three days and the tumor mass was taken out at the 30th day, the EGFR expression as well as GFAP were examined by immunohistochemical staining. The cell proliferating activity were examined by PCNA. Tumor samples were fixed and the expression of EGFR, PCNA and GFAP were evaluated by immunohistochemistry. R^ulls Lipofectamin mediated shRNA targeting EGFR dramatically down regulated its' expression in U251 glioma cells. Cell growth was inhibited by 68% as indicated by MTT assay. Decreased EGFR expression in shRNA transfected cells was accompanied by 12.7% to 13% decrease in U251 cells in S phase and 10.5% to 11.7% increase in G2 phase. In cell growth matrigel matrix, normal cell appearanced in parental ceils and psiRNA-scr transfected cells. While the cells transfected with shRNA were detached from the matrix or grew in a scattered clustering patterns, signified poor cell growth activities. The tumor volume of psiRNA-2400 treated group was smaller than that of the control (psiRNA-scr) and empty plasmid treated group ( P 〈 0.01 ). The effect of knocking down EGFR expression, the upregulation of GFAP, and in the inhibition of proliferative potential in psiRNA-2400 treated group were more significant than that in the two control groups. Conclusion shRNA targeting EGFR downregulates significantly its expression in a sequencespecific manner, exerting growth inhibition effect on U251 glioma cells in vitro and in vivo. Consequently, shRNA expressing plasmid mediated gene therapy may be a new strategy in targeting molecular therapy of gliomas.
出处
《中华神经外科杂志》
CSCD
北大核心
2007年第2期111-114,共4页
Chinese Journal of Neurosurgery
基金
国家自然科学基金资助项目(30400461)
天津市科委应用基础计划重点资助项目(05YFJZJC1002)