A172、U251及U251／VM-26的耐药性与鞘糖脂的关联研究

The relationship of the expression level of glycosphingolipids in human glioblastoma cell line A172, U251 and U251/VM-26 and drug resistance

目的研究人脑胶质瘤细胞系的耐药与鞘糖脂的相关性。方法采用MTT方法检测了人脑胶质瘤细胞系A172和U251对替尼泊苷的药物敏感性，然后质谱分析两株细胞系的鞘糖脂水平的变化，并采用逐渐增加药物浓度法建立U251细胞系耐替尼泊苷细胞系（U251／VM-26细胞系），然后MTr法检测其对替尼泊苷的敏感度、Real—timePCR技术检测其耐药基因MDRl和MRPl的表达、质谱分析细胞系耐药前后鞘糖脂的变化。结果MTr显示A172细胞对替尼泊苷的耐药性高于U251细胞，同时质谱分析发现A172细胞中C18-LacCer、C18-Gb3、C18．Gb4鞘糖脂表达明显高于U251细胞系。U251细胞系经诱导耐药后（U251／VM-26细胞系）其耐药性上升100倍（P〈0．001）；同时发现U251细胞系药物诱导耐药后C18-Gb4的表达上升，C18-LacCer和C18-Gb3鞘糖脂表达也上升。Real-timePCR显示A172细胞系中MRPl基因表达水平显著高于U251细胞系，U251／VM-26耐药细胞系中MRPl、MDRI基因显著高于U251细胞系。结论C18-LacCer、C18-Gb3和C18．Gb4鞘糖脂与A172、U251和U251／VM-26胶质瘤细胞系对替尼泊苷的敏感性相关，且耐药基因MDRl和MRPl参与了人脑胶质瘤细胞系对替尼泊苷的耐药。

Objective To study the relationship of the drug resistance and glycosphingolipids in glioma. Methods The sensitivity of the teniposide in A172 and U251 cells were detected by MTF, and their glycosphingolipids were detected by ESI-LIT-MS. The drug resistance cell line U251/VM-26 was established by culture with gradual increased dose of VM-26. Then the sensitivity of the teniposide in U251 and U251/VM-26 were checked by MTr, the drug resistance associated gene MDR1 and MRP1 were detected by real-time PCR, and their glycosphingolipids were detected by ESI-LIT-MS. Results The drug resistant of A172 were higher than U251 ceils. Meanwhile, C18-LacCer, C18-Gb3, C18-Gb~, are highly accumulated in A172 cells than in U251 cells. The drug resistant of U251/VM-26 cells was 100-folds higher than U251 ceils after co-cuturing with VM-26, then the levels of C18-Gb4 are significantly increased in U251/VM-26 cells, Cl8-LacCer and C18-Gb3 are also increased. Real-time PCR data show that MRP1 in A172 ceils are higher than U251 cells, the level of MDR1 and MRP1 gene are also highly accumulated in U251/VM-26 cells. Conclusions Glioma-associated glycosphingolipids, C18-LacCer,C18-Gb3 and C18- Gb4, are correlated with drug resistance of VM-26, and MDR1 and MRP1 gene are involved in the drug resistant of those glioma cells.