大剂量冲击法建立胶质瘤耐替莫唑胺细胞株及生物学鉴定

Development and characterization of temozolomide resistant glioma cell line by pulse exposure

目的:体外构建胶质瘤耐替莫唑胺细胞株,并探讨其生物学特征。方法:采用大剂量冲击法诱导构建人脑胶质瘤耐替莫唑胺(TMZ)U251细胞株(命名为U251/TMZ);采用MTT法检测亲代细胞(U251)和U251/TMZ对TMZ的敏感性;绘制细胞增殖倍增曲线,比较两种细胞生长情况;采用免疫荧光技术检测两者CD133的表达;蛋白质印迹法检测两种细胞ABCG2的表达。结果:成功构建胶质瘤耐替莫唑胺细胞株,其耐药指数为亲代的8.1倍(t=-63.28,P=0.00);U251/TMZ增殖倍增较亲代细胞延长;U251/TMZ细胞表达CD133较亲代细胞明显增加(t=45.35,P=0.00),U251/TMZ细胞ABCG2表达较亲代明显增高(t=6.19,P=0.00)。结论:通过大剂量冲击法构建的U251/TMZ细胞产生一定的耐药性,具有明显的胶质瘤干细胞生物学特性。

Objective： To build temozolomide resistant glioma cell line in vitro,and to investigate its biological characteristics.Methods： By using pulse exposure to induce and build resistant to temozolomide（TMZ） human glioma U251 cell lines（named U251/TMZ）;MTT was applied to detect the sensitivity of the parental cells（U251） and U251/TMZ to TMZ;drew proliferation curves of both cells and their growing conditions were compared;immunofluorescence technique was applied to detect CD133 expression;western blot assay was applied to detect the ABCG2 expression of both cells.Results： Temozolomide resistant glioma cell line had been successfully constructed and the resistance index of U251/TMZ was 8.1 times（t=-63.28,P=0.00） as high as parental cells;the proliferation U251/TMZ was slower than that of parental cells;by applying immunofluorescence assay to detect CD133 expression,there was a significant increase in U251/TMZ compared with parental cells（t=45.35,P=0.00）;by applying western blot assay to detect ABCG2 expression,it was found that U251/TMZ was significantly higher than pro-generation（t=6.19,P=0.00）.Conclusion： The U251/TMZ developed with the method of pulse exposure displayed a certain amount of resistance,and possessed obvious biological characteristics of glioma stem cells.