胶质瘤多药耐药细胞株U251/TMZ的生物学特性

目的探讨胶质瘤U251／替莫唑胺（TMZ）多药耐药细胞株的生物学特性。方法采用TMZ间歇浓度梯度递增法诱导建立胶质瘤多药耐药细胞株U251／TMZ，光镜观察细胞形态，细胞计数计算倍增时间，四甲基偶氮唑蓝法检测耐药指数，流式细胞仪检测细胞周期，逆转录-PCR法检测多药耐药性1（MDRl）、Bcl-2、多药耐药相关蛋白5（MRP5）、肺耐药相关蛋白1（LRPl）等mRNA表达。结果胶质瘤耐药细胞株U251／TMZ对TMZ、依托泊苷、硫酸长春新碱、环磷酰胺、阿霉素的耐药指数分别为4．39、3．61、2．64、2．00、1．63；细胞周期结果显示U251／TMZ细胞系较U251亲本细胞系G0/G1期和s期明显减少（P〈0．05），GfM期明显增多（P〈0．05）；U251／TMZ倍增时间（14．64＋1．98）h1较亲本细胞系u251（10．26＋1．03）h1明显延长（P〈0．05）；U251／TMZ耐药细胞株MDRl、Bcl-2、MRP5、LRPl等mRNA表达均较亲本细胞系U251明显上调。结论间歇TMZ浓度递增法可成功建立人脑胶质瘤U251多药耐药系，MDRl、Bcl-2、MRP5、LRPl的表达明显上调可能与耐药性形成有关。

Establishment and biological characteristics of multidrug- resistant glioma cell line U251/TMZ

Objective To establish in vitro the multidrug resistant glioma cell line, U251/temozolomide （TMZ）. Methods The different gradient concentration of TMZ acted on U251 cells. U251 cell morphology was observed by a light microscope. The time of doubling ceils was detected by the cell count technique. The U251/TMZ multidrug resistance was assayed by MTF. The changes in U251 cells and U251/TMZ cells cycles were detected by flow cytometry. RT-PCR assay resistant cell strains of the expressions of drug resistant genes including BCL-2, MRPS, LRP1 in U251 cells and U251/TMZ cells by RT-PCR assay. Results The multidrug-resistant glioma cell line, U251/VPTMZ cells was successfully established by intermittent concentration gradient increments technique. The drug-resistance of U251/VPTMZ to temozolomide, VP-16, vincristine sulfate, cyclophosphamide and doxorubicin increased respectively by were 361%, 1236%, 264%, 200% and 163%. The increases in the volumes of the cells bodies, which were pleomorphic, and had irregular shaped nucleus, and cytoplasmic granular substances, were observed in the multidrug- resistant cells. The cells in G0/G1 phase were significantly more and the cells in S phase significantly fewer in U251/TMZ than those in U251 cells （P〈0.05）. There was significant difference in the time of doubling cells between U251/TMZ and U251 （P〉0.05）. The expressions of MDR1, BCL-2, MRP5 and LRP1 in U251/TMZ in the multidrug resistant cell lines were significantly up regulated. Conclusions The muhidrug-resistant human glioma cells line with stable multidrug resistance may be successfully established by intermittent concentration gradient increment technique. Their stable multidrug resistance may be related to the up regulation of MDR1 BCL-2, MRP5 and LRP1 expressions.