细胞间P-糖蛋白转移介导膀胱癌BIU-87细胞形成稳定多药耐药的研究

发布时间：2018-07-03 10:18

本文选题：膀胱癌 + 多药耐药　；参考：《福建医科大学》2014年硕士论文

【摘要】：目的探讨细胞间P 糖蛋白转移现象在形成并维持膀胱癌BIU 87细胞多药耐药中的作用及机制。方法用Transwell共培养膀胱癌耐药株细胞BIU 87/ADM与敏感株细胞BIU 87，在共聚焦显微镜下观察共培养至24h、48h、72h、96h Transwell下室BIU 87细胞（即获得性耐药细胞AqMDR）P 糖蛋白含量，细胞计数法绘制BIU 87/ADM、BIU 87、共培养至96h AqMDR三种在加与不加1μg/ml阿霉素（adriamycin,ADM）培养液中的生长曲线并比较其倍增时间，分离出共培养至96h AqMDR继续分2组传代培养，一组培养基中加入1μg/ml阿霉素，另一组不加。分别运用CCK 8、Western Blot、RT PCR方法、流式细胞仪检测0、4、8、16、20代及第20代冻存1个月后复苏的AqMDR的耐药指数、P gp表达量、MDR1mRNA表达水平及细胞内罗丹明 123荧光强度。结果共聚焦显微镜结果显示P 糖蛋白转移量随着共培养时间延长逐渐增多。细胞生长曲线提示：在不加ADM组，BIU 87倍增时间（25.30+0.04h）较BIU 87/ADM（31.58+0.37h）短（P0.001），AqMDR介于两者之间（28.39+0.33h，P0.001）；在加入1μg/ml ADM组，BIU 87培养5天后全部死亡，而AqMDR与BIU 87/ADM细胞生长并不受限制。CCK 8、Western Blot、RT PCR及罗丹明 123外排实验证实：AqMDR细胞随着传代增加，在不加阿霉素组，耐药指数和P gp表达量逐渐下降，细胞内罗丹明 123荧光强度逐渐升高，最终回到敏感株BIU 87水平，，MDR1mRNA的表达水平未见明显改变。在加阿霉素组，耐药指数和P gp表达量略呈增高趋势，细胞内罗丹明 123荧光强度略呈下降趋势，MDR1mRNA的表达水平逐渐增高到耐药株细胞BIU 87/ADM水平。结论细胞间P 糖蛋白转移量随着耐药株与敏感株共培养时间延长而逐渐增多，AqMDR细胞能够在略大于敏感株IC50的药物浓度（此浓度会导致敏感株死亡）下稳定生长，细胞间P gp转移有利于膀胱癌敏感细胞逃避化疗药物的作用并由暂时性耐药发展为获得性永久耐药。
[Abstract]:Objective to investigate the role and mechanism of intercellular P glycoprotein transfer in the formation and maintenance of multidrug resistance (MDR) in bladder cancer cells. Methods Transwell co-cultured bladder cancer cell line BIU / 87 / ADM and sensitive cell line BIU / 87 / ADM were used to observe the content of P-glucoprotein (P) in BIU _ (87) cells (namely, acquired drug resistant cells AqMDR) under confocal microscope to 24 h ~ (48) h ~ (72) h ~ 96 h. The cell count method was used to draw the growth curve of three kinds of AMDR in medium supplemented or without 1 渭 g/ml adriamycin (ADM) for 96 h, and the doubling time was compared. The co-cultured cells were isolated and cultured to 96 h, AqMDR was further subcultured in two groups, and the results were as follows: 1 渭 g/ml adriamycin (adriamycin) adriamycin was added to the culture medium, and the time of multiplication was compared. In one group, 1 渭 g/ml adriamycin was added to the medium, while in the other group, no adriamycin was added. The expression level of MDR1 mRNA and the fluorescence intensity of Neronerol 123 were detected by flow cytometry and flow cytometry, respectively. The drug resistance index (MDR) and the expression level of MDR1 mRNA were detected by flow cytometry after cryopreservation for 1 month or 20 months after cryopreservation for 1 month. The expression of MDR1 mRNA and the fluorescence intensity of Nerosin 123 in cells were detected by flow cytometry (FCM). The expression level of MDR1 mRNA and the expression level of MDR1 mRNA were measured. Results the results of confocal microscopy showed that the amount of P glycoprotein transfer increased with the prolongation of co culture time. The cell growth curve indicated that the doubling time of BIU87 (25.300.04 h) was shorter than that of BIU-87 / ADM (31.58 0.37 h) in the group without ADM (P0.001). However, the growth of AqMDR and Biu 87 / ADM cells was not restricted. The results of Western Blot RT PCR and Rhodamine 123 efflux assay showed that the drug resistance index and the expression of P P in the group without adriamycin decreased gradually with the increase of passage. The fluorescence intensity of Nero-danmycin 123 increased gradually, and the expression level of MDR1 mRNA in the sensitive strain BIU-87 did not change significantly. In the adriamycin group, the drug resistance index and the expression of P ~ + GP increased slightly, and the fluorescence intensity of Nero-danmycin 123 decreased slightly. The expression level of MDR1 mRNA increased gradually to 87% ADM level in the resistant cell line. Conclusion with the increase of co-culture time between resistant strain and sensitive strain, the amount of P-glucoprotein transfer between cells increased gradually, and the cells of AqMDR could grow stably at a concentration slightly larger than that of sensitive strain IC50 (which would lead to the death of sensitive strain). Intercellular P P GP metastasis was beneficial to the escape of chemotherapeutic agents from transient resistance to acquired permanent resistance in bladder cancer sensitive cells.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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