核转录因子NF-κB及黏蛋白MUC1参与膀胱癌耐药机理的研究

Study of drug resistance mechanism of NF-κB and MUC1 in bladder cancer cell lines

目的探讨核转录因子NF-κB及上皮特异性黏蛋白MUC1基因及蛋白参与膀胱癌细胞耐药形成的机理。方法免疫组织化学SP法检测NF-κB p65和MUC1蛋白在B IU-87、T-24及耐药株B IU-87/A细胞中的表达。原位杂交技术检测NF-κB p65的mRNA在3种细胞质中的表达。流式细胞仪检测3种细胞在阿霉素、长春新碱、顺铂3种化疗药物作用48 h后的凋亡率。结果NF-κB p65蛋白在B IU-87、T-24细胞质中均有阳性表达,细胞核几乎无表达,在B IU-87/A耐药株的胞质、胞核中均有阳性表达。3组细胞平均吸光度值差异无统计学意义(P>0.05)。MUC1在B IU-87、T-24细胞膜、胞质中均有弱阳性表达,在B IU-87/A的胞膜、胞质中均有强阳性表达,两者平均吸光度值差异有统计学意义(P<0.01)。3种细胞中NF-κB p65 mRNA表达均在胞质,核无表达,且3组细胞平均吸光度值差异无统计学意义(P>0.05)。3种细胞自发凋亡率分别为1.15%、1.40%、0.90%,3种化疗药物作用48 h后,B IU-87细胞凋亡率分别为45.69%、47.70%、44.50%,T-24细胞凋亡率分别为43.79%、46.17%、44.50%,用药前后差异有统计学意义(P<0.01)。两种细胞之间及3种化疗药之间差异无统计学意义(P>0.05)。B IU-87/A在阿霉素、长春新碱作用48 h后的凋亡率分别为19.88%、21.41%,用药前后差异有统计学意义(P<0.05)。结论NF-κB是膀胱癌细胞B IU-87/A耐药形成的中心环节,B IU-87/A耐药形成中NF-κB过度活化导致MUC1高表达可能是另一途径。

Objective To investigate the drug resistance mechanism of NF-κB and MUC1 in bladder cancer cell lines. Methods The expressions of NF-κB P65 and MUC1 were detected in BIU-87 cells, T-24 ceils and BIU-87/A drug resistant cells by immunohistochemistry SP method. The expression of NF-κB P65 mRNA was detected in BIU-87 ,T-24 and BIU-87/A drug resistant cells by hybridization in situ. Apoptosis rates of these 3 cells were detected by flow cytometry after adriamycin,vincristine and cisplatin cytotoxic drug acting for 48h. Results NF-κB P65 protein was expressed only in cytoplasm of BIU-87 and T-24 cells,and expressed in both cytoplasm and cytoblast of BIU-87/A drug resistant cells. There was no significant difference of the mean values of absorbance among the 3 cells （ P 〉 0.05 ）. MUC1 was expressed weakly in cytoplasm and membrane of BIU-87 and T-24 cells,while it was expressed strongly in cytoplasm and mem- brane of BIU-87/A drug resistant cells,with significant difference between sensitive cells and resistant cells （ P 〈 0.01 ）. NF-κB p65 mRNA of the 3 ceils was expressed only in cytoplasm （ not in nucleus） with no significant difference of the mean values of absorbance among them （ P 〉 0.05 ）. Apoptosis rates of BIU-87, T-24 and BIU-87/A cells were 1.15% , 1.40% and 0. 90%. The rates of BIU-87 cells were 45.69% ,47.70% and 44.50% ;the rates of T-24 cells were 43.79% ,46.17% and 44.50% after adriamycin, vincristine and cisplatin cytotoxic drugs acting for 48 h. There were significant differences of the rates before and after use of these drugs （P 〈 O. 01 ）. However, the difference was not significant among the 3 cytotoxic drugs and between the 2 cells （P 〉 0. 05 ）. Apoptosis rates of BIU-87/A cells were 19.88% and 21.41% after adriamycin and vincristine acting for 48 h, with significant difference of the rates before and after use of the drugs （ P 〈 0. 05）. Conclusions NF-κB is an important central link of drug resistance mechanism of BIU-87/A ceils. Overexpression of MUC1 in drug resistant cells is a downstream event of active NF-κB and another drug resistance mechanism besides anti-apoptosis path and multidrug resistance gene. Drug resistance mechanism is completed by multigene and muhipath. Flow cytometry for detecting apoptosis rate is an early,sensitive and reliable method for evaluation of drug resistance in bladder cancer cell lines.