U266/BOR耐药细胞株的构建及其生物学活性鉴定

Establishment of BOR-Resistant U266 Cell Line and the Detection of Its Biological Activities

目的:建立人对硼替佐米(BOR)耐药的多发性骨髓瘤U266细胞株(U266/BOR),并检测其生物学特性。方法:应用小剂量诱导和剂量递增联合诱导法建立耐药细胞株U266/BOR;在倒置显微镜下观察细胞的形态;应用MTT法测定两种细胞的BOR半数抑制浓度(IC50)和耐药系数;绘制两种细胞的生长曲线并计算二者的倍增时间;流式细胞术检测两种细胞的细胞周期,RT-PCR法检测两种细胞的相关耐药基因mRNA水平。结果:成功建立了耐药系数为19.8的耐药细胞系U266/BOR;二者细胞形态上未见明显差别;U266/BOR较U266细胞生长缓慢,其G_0/G_1期比例增加,S期比例减少;U266/BOR细胞中耐药基因PTPROt、Beclin 1及PTEN的mRNA表达减少,而c-Maf的mRNA含量增加,但MDR1的mRNA表达量无明显差别。结论:成功构建人对BOR耐药的MM细胞株U266/BOR,为BOR耐药机制的深入研究提供了理想细胞模型。

Objective： To establish bortezomib（ BOR）-resistant human multiple myeloma U266 cell line U266/BOR and to detect its biological characteristics. Methods： U266 cells were constantly exposed at low dose and progressively increasing dose of BOR to establish U266/BOR,the cell morphology was observed by inverted microscopy,IC50 and resistant index were determined by MTT assay,cell growth curve was drawed and the doubling time was calculated; cell cycle distribution were analyzed by flow cytometry,and RT-PCR was used to detect the mRNA expression of resistancerelated genes. Results： The MM U266/BOR cell line was successfully constructed and its resistance index was up to 19. 8.The both cell morphologies were not different. Compared with U266 cells,the multiplication time was postponed with the increase of G0/G1 cell ratio,and S phase was reduced. The mRNA expression of PTPROt,Beclin 1 and PTEN were reduced,and the mRNA expression of c-Maf was enhanced in U266/BOR cells; as compared with U266 cells,but the MDR1 mRNA expression was not different between U266 cells and U266 BOR cells. Conclusion： The BOR-resistant U266 cell line has been establiseed successfully. It provides an ideal cell model for further exploration of the mechanism for BOR resistance.