反义GFAP对人恶性胶质瘤细胞系CHG-5增殖及分化的影响

目的观察抑制内源性胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)基因表达对恶性胶质瘤细胞生物学特性的影响,为进一步深入研究GFAP基因在胶质瘤恶性转化及诱导分化治疗中的作用提供实验基础.方法构建反义GFAP 逆转录病毒表达载体,经包装后以病毒上清感染人恶性胶质瘤细胞系CHG-5;采用RT-PCR、原位杂交以及免疫细胞化学方法检测GFAP基因及其蛋白的表达;并通过形态学、细胞生长曲线、软琼脂克隆形成及流式细胞分析等观察抑制GFAP基因表达后CHG-5细胞形态、增殖和细胞周期进程的变化.结果反义逆病毒感染后CHG-5细胞GFAP mRNA及其蛋白表达显著减弱甚至缺失,瘤细胞异型性增加,细胞增殖速度加快,体外成瘤能力增强,S期、G2/M期细胞比例升高.结论抑制内源性GFAP基因表达可使CHG-5细胞呈现更为恶性的表型,提示GFAP基因在调控胶质瘤细胞分化程度及恶性生物学行为方面具有重要作用.

Effects of antisense GFAP retrovirus on cell proliferation and differentiation of human glioma cell line

Objective To observe the effects of suppression glial fibrillary acidic protein (GFAP) gene expression on the biological features of human malignant glioma cells in vitro . Methods A recombinant retrovirus expression vector containing 1.1 Kb GFAP cDNA in the antisense orientation was constructed. The human glioma cell line, CHG 5, was infected with the suspension of this retrovirus after its packing and concentrating. RT PCR, in situ hybridization as well as immunocytochemical method were employed to identify the expression of GFAP gene and its protein. The infected and control glioma cells were analyzed for the alterations of cell cycle progression, morphology, and cell growth features by using flowcytometry, cloning formation on soft agar and other methods. Results The expression of GFAP mRNA and its protein were reduced or even eliminated in CHG 5 cells infected with antisense GFAP retrovirus. In contrast to the control, GFAP negative CHG 5 cells demonstrated profound morphological alterations and marked cellular atypia. Cell proliferation rate and their tumorigenecity in vitro were also enhanced. Cell cycle analysis results revealed that cell proportion of S and G 2/M phases was increased significantly. Conclusion These results illustrate that suppression GFAP gene expression has affected CHG 5 glioma cell morphology, and enhanced their growth and tumorigenesity. The data also suggest that GFAP gene might play a key role in determination of differentiation and certain malignant biological features of glioma cells.