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目的:观察miR-218对胶质母细胞瘤细胞系成熟分化的影响。方法:通过质粒转染和G418筛选建立稳定过表达miR-218的SNB-19亚细胞系及对照亚细胞系。利用qRT-PCR及Western blot检测稳定细胞系中miR-218、CD133及Nestin表达水平。用GFAP免疫细胞化学染色评价其成熟分化水平,通过免疫荧光观察转染miR-218表达质粒后CD133及Nestin阳性细胞的分布情况。结果:过表达miR-218的亚细胞系中miR-218、CD133及Nestin表达水平分别是对照细胞的26.23倍、17.9%及54.2%,过表达miR-218细胞的GFAP阳性标记指数为(96.0±3.81)%显著高于对照细胞(49.6±5.13)%(t=16.24,P<0.0)1)。免疫荧光显示CD133及Nestin的下调特异性发生于成功转染miR-218表达质粒的细胞。结论:miR-218可通过促进胶质瘤干细胞的成熟分化显著下调CD133及Nestin的表达水平,该作用可能是miR-218抑制胶质瘤细胞增殖的重要机制之一。
Objective: To observe the effect of miR-218 on the maturation and differentiation of glioblastoma cell lines. Methods: SNB-19 sub-cell line and control sub-cell line stably overexpressing miR-218 were established by plasmid transfection and G418 selection. The expression of miR-218, CD133 and Nestin in stable cell lines was detected by qRT-PCR and Western blot. GFAP immunocytochemistry was used to evaluate the maturation and differentiation of CD133 and Nestin-positive cells. The expression of miR-218 was detected by immunofluorescence. Results: The expression of miR-218, CD133 and Nestin in sub-lines overexpressing miR-218 were 26.23 folds, 17.9% and 54.2% compared with that of control cells, respectively. The GFAP positive labeling index of miR-218 cells overexpressing was (96.0 ± 3.81%) was significantly higher than the control cells (49.6 ± 5.13)% (t = 16.24, P <0.0) 1). Immunofluorescence showed that the down-regulation of CD133 and Nestin occurred in cells successfully transfected with miR-218 expression plasmid. Conclusion: miR-218 can significantly down-regulate the expression of CD133 and Nestin by promoting the differentiation of glioma stem cells, which may be one of the important mechanisms by which miR-218 inhibits the proliferation of glioma cells.