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目的探讨B7家族分子B7-H6在脑胶质瘤干细胞样细胞(GSLC)中的表达及其意义。方法使用亚克隆的方法并利用GSLC在体外形成神经细胞球的特性,从U87细胞株中筛选得到脑GSLC;通过实时定量PCR和流式细胞术检测干细胞标志分子c-myc、性别决定区基因Y盒2(Sox2)、CD133、神经上皮干细胞蛋白(nestin)、CXC趋化因子受体4(CXCR4)的表达;通过化疗药物多柔比星、顺铂、卡铂杀伤鉴定其是否具有耐药特性;通过实时定量PCR及流式细胞术获得脑GSLC中B7家族的表达;采用小干涉RNA(si RNA)干扰下调B7-H6表达,CCK-8法检测细胞增殖的变化。结果使用亚克隆方法分离获得的脑GSLC能在体外形成神经细胞球,上调表达多种干细胞标志分子CD133、nestin、CXCR4,具有显著的耐药特性。B7家族分子B7-H1、B7-H3、B7-H4和B7-H6在得到脑GSLC中均表达,与原代U87细胞相比,细胞膜上的B7-H6的表达发生显著上调,使用si RNA干扰B7-H6表达后,细胞的增殖受到抑制,且c-myc基因的表达也下调。结论 U87细胞来源的脑GSLCB7-H6表达上调,并且与GSLC生物学特性相关。
Objective To investigate the expression of B7-H6 in glioma stem cell-like cells (GSLC) and its significance. Methods The subcellular localization of c-myc was determined by real-time quantitative PCR and flow cytometry. The gene of sex determination region Y (Sox2), CD133, nestin, and CXC chemokine receptor 4 (CXCR4) expression were evaluated by chemotherapeutic drugs doxorubicin, cisplatin and carboplatin The expression of B7 family in brain GSLC was detected by real-time PCR and flow cytometry. The expression of B7-H6 was down-regulated by si RNA interference and the proliferation was detected by CCK-8. Results The brain GSLC isolated by subcloning method could form neurospheres in vitro and upregulate the expression of various stem cell marker molecules CD133, nestin and CXCR4, and had significant drug resistance. B7-H1, B7-H3, B7-H4 and B7-H6 were all expressed in brain GSLC. Compared with primary U87 cells, the expression of B7-H6 on B7-H6 cells was significantly up-regulated. After B7-H6 expression, cell proliferation was inhibited, and c-myc gene expression was also down-regulated. Conclusion U87 cell-derived brain GSLCB7-H6 expression is up-regulated and is related to the biological characteristics of GSLC.