摘要
目的 培养胶质母细胞瘤干细胞,观察microRNA（miR）-153对其生物学功能的影响,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）,分化后细胞神经胶质纤维酸性蛋白（GFAP）、微管相关蛋白2（MAP2）的表达进行鉴定.RT-PCR检测分选后CD133＋细胞、CD133-细胞miR-153的表达;将人miR-153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-153、NC组,7d后用成球试验检测miR-153对胶质母细胞瘤干细胞自我更新能力的影响.RT-PCR、免疫荧光染色分别检测2组细胞CD133、nestin、GFAP、MAP2 mRNA和蛋白的表达.转染后24、48、72、96和120 h CCK-8法检测2组细胞存活率.转染后3d流式细胞仪检测2组细胞凋亡率. 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;分选后CD133＋细胞中miR-153的表达低于CD133-细胞,差异有统计学意义（P＜0.05）.转染后7 dNC组细胞一、二次成球数目均高于miR-153组,差异有统计学意义（P＜0.05）; RT-PCR检测显示与NC组比较,miR-153组细胞CD133、nestin mRNA表达降低,GFAP、MAP2 mRNA表达升高,差异有统计学意义（P＜0.05）.免疫荧光染色与RT-PCR检测结果一致;转染后48、72、96 h miR-153组的细胞存活率均低于NC组,差异均有统计学意义;流式细胞仪检测显示miR-153组的细胞凋亡率（9.41％±1.98％）高于NC组（4.28％±0.31％）,差异有统计学意义（P＜0.05）. 结论 上调miR-153可能成为新的针对胶质母细胞瘤干细胞的治疗策略.
Objective To isolate the glioblastoma multiforme stem cells （GBM-SCs） from GBM specimens and to investigate the biological role ofmiR-153 in GBM-SCs so as to explore the application of gene therapy of GBMs.Methods CD133＋ cells were separated using magnetic cell sorting technique （MACS） after primary culture.Immunofluorescence staining was employed to detect the CD133,nestin,glial fibrillary acidic protein （GFAP）,microtubule-associated protein 2 （MAP2） expressions; real time-PCR was used to analyze miR-153 mRNA expression in CD 133-cells and CD 133＋ cells.Lipofectamine RNAiMAX was used to transfect MiR-153 mimic （miR-153 group） and scrambled control oligonucleotides （NC group） into GBM-SCs; 7 d after that,sphere formation assay was performed to determine the self-renewal ability of GBM-SCs.Real time-PCR and immunofluorescence were carried out to examine the CD133,nestin,GFAP,MAP2 mRNA and protein expressions.At last,the proliferation ability of miR-153 treated GBM-SCs and NC cells was determined by CCK-8 at 24,48,72,96 and 120 h after the transfection and the apoptosis ratio was detected by flow cytometry 3 d after the transfection.Results GBM-SCs isolated from GBM specimens could express stem cell markers CD133 and nestin; after differentiation,the cells could express astrocyte marker GFAP and neuron marker MAP2; miR-153 expression in CD133＋ cells was signficantly down-regulated as compared with that in CD133-cells （P〈0.05）.Seven d after transfection,the number of spheres in the NC group was significantly larger than that in the miR-153 group （P〈0.05）; real time-PCR indicated that the mRNA expressions of CD 133 and nestin in the miR-153 group were significantly decreased,and the GFAP and MAP2 mRNA expressions were statistically increased as compared with those in the NC group （P〈0.05）.The results detected by immunofluorescence were in accordance with those by real time-PCR; 48,72,96h after the transfection,cell viability in cells from miR-153 group was statistically
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2015年第3期233-238,共6页
Chinese Journal of Neuromedicine
基金
广东省医学科研基金（B2014417）
惠州市科技计划项目（20140802）
