摘要目的 培养胶质母细胞瘤干细胞,观察微小RNA (miR)-203对其干细胞的影响,探讨其在胶质母细胞瘤基因治疗中的应用.方法 体外原代培养胶质母细胞瘤组织,免疫磁珠法分选CD133+细胞;免疫荧光染色检测分选后细胞CD133、巢蛋白(Nestin)、神经胶质纤维酸性蛋白(GFAP)、微管相关蛋白2(MAP2)的表达.将人miR-203模拟体和无意义寡核苷酸链(NC)分别转染胶质母细胞瘤干细胞作为miR-203组、NC组,用成球试验检测miR-203对胶质母细胞瘤干细胞自我更新能力的影响.反转录-聚合酶链反应(RT-PCR)、免疫荧光染色分别检测两组细胞CD133、Nestin、GFAP、MAP2的mRNA和蛋白的表达.结果 培养的胶质母细胞瘤干细胞表达干细胞标记物CD133、Nestin,分化后细胞表达星形胶质细胞的标志物GFAP、神经元的标志物MAP2;转染后7d,当NC组细胞球数目为100％时,miR-203组一、二次球数目分别为(67.84±13.79)％和(30.53±8.91)％,差异均有统计学意义(P＜0.05);RT-PCR结果显示与NC组比较,miR-203组细胞CD133mRNA的相对表达水平为0.29±0.15,nestin mRNA为0.27±0.18;GFAP、MAP2mRNA表达升高,分别为7.89±3.99和2.59±1.16,差异有统计学意义(P＜0.05).结论 miR-203可以抑制胶质母细胞瘤干细胞的自我更新能力并促进其多向分化,可能成为新的针对胶质母细胞瘤的治疗策略.

abstractsObjective To isolate glioblastoma multiforme (GBM) stem cells (GBM-SCs) from GBM specimens and to investigate the biological role of miR-203 in GBM-SCs' stemness properties.Methods CD133 + cells were separated using magnetic cell sorting technique (MACS) after primary culture.Lipofectamine RNAiMAX was used to transfect miR-203 mimic and scrambled control oligonucleotides into GBM-SCs.To determine the self-renewal ability of GBM-SCs,we performed sphere formation assays.Then,reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence analysis were carried out to examine the expression level of stemness and differentiation markers.Results GBM-SCs isolated from GBM specimens formed GBM spheres,expressed markers associated with neural stem cells,and possessed the capacity for self-renewal and multilineage differentiation.In primary and secondary sphere formation assays,when the number spheres in the control group was assumed to be 100％,only (67.84 ± 13.79)％ and (30.53 ±8.91)％ formed from miR-203 transfected group.After 3 days of transfection,we assayed the mRNA levels of CD133,nestin,GFAP and MAP2 using qRT-PCR analysis,the fold changes of these markers were 0.29 s0.15,0.27 ±0.18,7.89 ±3.99 and 2.59 ±1.16.Then we used fluorescence microscope to detect changes in the protein levels.In miR-203 transfected group,the expression of CD133 and Nestin were obviously weaker than in the control group,and the expression levels of GFAP and MAP2 were stronger.Conclusion Reactivation of miR-203 expression suggests novel therapeutic strategies for GBM-SCs.