体外培养脑胶质瘤细胞株中CD133表达的研究

Expression of CD133 in in vitro cultured glioma cells

目的探讨与脑胶质瘤干细胞相关的CD133表型。方法将无血清培养中的胶质瘤干细胞与含血清贴壁培养的胶质瘤细胞中CD133的表达进行对比。逆转录．聚合酶链反应（RT-PCR）及实时荧光定量PCR检测信使RNA（mRNA）水平的表达，流式细胞术及免疫荧光染色法检测蛋白水平的表达，激光共聚焦扫描成像进行CDl33蛋白的亚细胞定位。结果所有胶质瘤细胞中均存在CD133mRNA及蛋白的表达。AC133抗体所识别的糖基化CD133-AC133分子仅分布于无血清培养中的胶质瘤干细胞，阳性细胞比例为31．8％～99．9％，且定位于此类细胞膜表面。AC133阴性的胶质瘤干细胞及含血清贴壁培养胶质瘤细胞中CD133蛋白的表达位于细胞内，阳性细胞比例约为22．2％～88．6％，其亚细胞定位为内质网和／或高尔基体。结论细胞表面AC133分子，而非CD133mRNA及细胞内CD133蛋白标记了一类胶质瘤干细胞亚群，但无血清培养环境下也存在AC133呈阴性表达的胶质瘤干细胞亚群。

Objective To discuss the CD133 phenotype correlating with glioma stem cells. Methods The expression of CD133 in glioma stem cell lines which were cultured in stem cell permissive serum-free medium was compared with that in conventional adherent glioma cell lines which were established in serum-containing medium. Reverse transcription-polymerase chain reaction （RT-PCR） and real-time quan-titative PCR were performed to study the expression level of CD133 mRNA. Flow cytometry and immunoflu-orescent staining were done to study the protein expression. Con_focal microscopy was adopted to study the subcellular localization of CD133 protein. Results CD133 mRNA and protein were distributed both in glioma stem cells and conventional adherent glioma cell lines. The glycosylated CD133-AC133 detected by AC133 antibody was only found in glioma stem cells cultured in serum-free medium and located on the cell membrane, and the proportion of positive cells was 31.8%-99. 9%. The CD133 protein in the AC133 neg-ative glioma stem cells and conventional adherent glioma cell lines was found intracellularly and located in the endoplasmic reticulum and/or Golgi apparatus, and the proportion of positive cells was 22. 2%- 88. 6%. Conclusion Neither CD133 mRNA nor intracellular CD133 protein but AC133 phenotype on the cell membrane is closely correlated with glioma stem cells, but a group of AC133 negative glioma stem cells can also exist in serum-free medium.