反义表皮生长因子受体cDNA转染抑制胶质母细胞瘤生长的机制研究

Mechanism of antisense epidermal growth factor receptor cDNA in growth suppression of glioblastomas cells

目的 探讨反义表皮生长因子受体(EGFR)cDNA转染抑制胶质母细胞瘤细胞株U87MG生长的机制。方法 反义EGFR cDNA转染胶质母细胞瘤细胞株U87MG后,筛选出低表达EGFR的细胞株;通过观察肿瘤细胞的形态和Western蛋白印迹免疫化学方法检测胶质纤维酸性蛋白(GFAP)的表达,来研究反义EGFR cDNA转染对胶质母细胞瘤分化的影响;通过流式细胞仪进行细胞周期分析及免疫组织化学方法检测细胞周期调控蛋白p53、Rb、p16和CDK4等,以研究反义EGFRcDNA转染对细胞周期的影响及机制;用telomeric repeat amplification protocol(TRA分析)检测肿瘤细胞的端粒酶活性。结果 反义EGFR cDNA转染胶质母细胞瘤U87MG后,肿瘤细胞的突起延长,细胞的GFAP表达增高;肿瘤细胞的G_0/G_1期细胞百分率明显增高,而S期细胞百分率明显减少;肿瘤细胞的野生型p53蛋白表达明显增高,而Rb、p16和CDK4等的蛋白表达水平未发生明显改变;肿瘤细胞的端粒酶活性明显降低。结论 反义EGFR cDNA转染可以通过诱导胶质母细胞瘤细胞分化、诱导野生型p53的表达、G_1细胞周期阻滞及抑制端粒酶活性等机制而抑制肿瘤细胞生长,而且这些作用机制之间是相互作用、相互协调而共同发挥抑制肿瘤细胞生长作用的。

Objective To study the mechanism of antisense epidermal growth factor receptor cDNA in growth suppression of glioblastomas cells. Methods Glioblastoma U87MG cells, which over-express epidermal growth factor receptor (EGFR) , were transfected with antisense-EGFR constructs. Several clones with stable expression of lower or undetectable levels of EGFR protein were obtained. The effect of antisense-EGFR on cell differentiation was studied using morphological evaluation and western blotting analysis of glial fibrillary acidic protein ( GFAP) expression. The effect of antisense-EGFR on cell cycle was studied by flow cytometry and immunohistochemical analysis of p53, Rb, p16 and CDK4 expressions. The effect of antisense-EGFR on telomerase activity was studied by telomeric repeat amplification protocol (TRAP) assay. Results U87MG cells that were transfected with antisense-EGFR constructs had smaller cell bodies and longer processes, and expressed higher level of GFAP compared with that of the control cells. Flow cytometric analysis showed that the proportion of cells in G0/G1 phases of the cell cycle in the antisense EGFR cDNA transfected clones increased significantly when compared with control cells, whereas the proportion of cells in S phase decreased markedly. In addition, immunohistochemical analysis showed that the expression of wild-type p53 was significantly increased in the antisense-EGFR cDNA transfected clones, whereas the expressions of Rb, p16 and CDK4 were not altered. TRAP assay revealed that telomerase activity in the antisense-EGFR clones was significantly decreased. Conclusions Antisense-EGFR transfection inhibits U87MG cell growth by inducing cell differentiation and p53 expression, G1 cell cycle arrest and inhibition of telomerase activity.