中文摘要：

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Westernblot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〈0．01）。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。

英文摘要：

Objective： To investigate the stimulatory effects of miR-218 on the differentiation of glioblastoma cells. Methods： The SNB-19 glioblastoma subcell line overexpressing miR-218 （ SNB19-miR218 ） and the control subcell line overexpressing a non- sense scrambled sequence （ SNB19-scr ） were established via plasmid transfection and G418 screening. The expression levels of miR-218, CD133, and nestin were detected via quantitative reverse transcription polymerase chain reaction and western blot analysis, whereas the differentiation status was assessed via glial fibrillary acidic protein （ GFAP ） immunocytochemistry. Using low-copy plas- mid transfected cells, the distribution of CD133- and nestin-positive cells was detected via immunofluorescence against an enhanced green fluorescent protein （ EGFP ） reporter. Results： The expression levels of miR-218, CD 133, and nestin in the cells transfected with SNBI9-miR218 were 26.23-fold, 17.9%, and 54.2% of those of SNB19-scr. The GFAP labeling index of SNB19-miR218 （ 96.0%± 3.81% ） was significantly higher than that of SNB19-scr （ 49.6% ±5.13%, t = 16.24, P 〈 0.0001 ）. Immunofluorescence showed that the constituent ratios of the CD133- and nestin-positive cells in the EGFP （ ＋ ） and （ - ） subpopulations were significantly uneven （ X 2 = 586.38 and 658.53, respectively, P 〈 0.0001 ）, confirming that the CD133 and nestin were downregulated specifically in the tumor cells successfully transfected with the miR-218 expression plasmid. Conclusion： miR-218 promotes glioma stem cell differentiation, and therefore downregulates CD133 and nestin expression. This effect may be an important mechanism for inhibiting the proliferation of glioma cells.