微小RNA-203抑制胶质母细胞瘤干细胞的干细胞特性  

MicroRNA - 203 can inhibit stenmess properties of glioblastoma multiforme stem cells

作　　者：邓一帆[1] 祝刚[1] 黄小山[1] 李百升[1] 秦忠宗[1] 

机构地区：[1]惠州市中心人民医院神经外科,广东516001

出　　处：《中华实验外科杂志》2016年第3期675-677,共3页Chinese Journal of Experimental Surgery

基　　金：广东省医学科研基金（B2014417）;惠州市科技计划（20140802）

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士0．15，nestinmRNA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。Objective To isolate glioblastoma multiforme （GBM） stem cells （GBM -SCs） from GBM specimens and to investigate the biological role of miR - 203 in GBM - SCs＇ sternness properties. Methods CD133 ± cells were separated using magnetic cell sorting technique （MACS） after primary cul- ture. Lipofectamine RNAiMAX was used to transfect miR -203 mimic and scrambled control oligonucleoti- des 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 a- nalysis 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 ± 0. 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 trans- fected 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.

关 键 词：胶质母细胞瘤 胶质母细胞瘤干细胞 微小RNA-203 自我更新 多向分化 

分 类 号：R730.264[医药卫生—肿瘤]