论文部分内容阅读
目的:建立白血病耐药细胞系U937/ADR模型,并检测其多药耐药相关基因及其生物学性状的改变。方法:以大剂量阿霉素(IC50浓度),短时间(2h)暴露法诱导人白血病细胞系U937细胞的阿霉素耐药性。检测细胞的生长曲线,计算阿霉素耐药倍数,流式细胞术分析细胞周期分布;罗丹明123检测药物外排功能;荧光定量PCR(FQ-PCR)检测MDR1、MRP1、NF-Κb、Bcl-2、BaxmRNA水平变化;Western blot检测Akt、p-Akt、P65、P-gp、MRP1和Bcl-2蛋白水平变化。结果:成功构建耐阿霉素U937/ADR细胞系,对阿霉素耐药指数为亲代U937细胞的11倍,U937/ADR群体倍增时间为43.6h,高于亲代细胞8.9h;流式细胞分析显示与U937细胞相比,U937/ADR的G0/G1期细胞增多,而G2/M期细胞减少。并对多种化疗药物产生交叉耐药性。罗丹明123外排试验显示,U937/ADR细胞外排明显增加。U937/ADR细胞MDR1、NF-Κb、Bcl-2mRNA表达水平明显增加,P-gp及p-Akt、P65表达水平增加。结论:成功构建的U937/ADR细胞系其生物学特性明显不同与亲代U937细胞,对多种化疗药物产生多药耐药,高表达多药耐药蛋白P-gp,同时激活p-Akt及NF-Kb。
OBJECTIVE: To establish a leukemic cell line U937 / ADR model and to detect the changes of multidrug resistance related genes and their biological characters. Methods: The doxorubicin resistance of human leukemia cell line U937 cells was induced by high dose of doxorubicin (IC50) and short time (2h) exposure. The cell growth curve was measured, the doxorubicin resistance rate was calculated, and the cell cycle distribution was analyzed by flow cytometry. The drug efflux function was detected by rhodamine 123. The expression of MDR1, MRP1, NF-κB, Bcl- -2, Bax mRNA levels were detected by Western blot. The protein levels of Akt, p-Akt, P65, P-gp, MRP1 and Bcl-2 were detected by Western blot. RESULTS: The ADR-resistant U937 / ADR cell line was successfully constructed, the resistance index to doxorubicin was eleven times higher than that of U937 cells, and the doubling time of U937 / ADR population was 43.6h, which was higher than that of parental cells 8.9h. Flow cytometry Compared with U937 cells, U937 / ADR cells showed increased G0 / G1 phase cells and G2 / M phase cells decreased. And a variety of chemotherapy drugs cross-resistance. Rhodamine 123 efflux assay showed significantly increased U937 / ADR cell efflux. U937 / ADR cells MDR1, NF-κB, Bcl-2mRNA expression increased significantly, P-gp and p-Akt, P65 expression increased. Conclusion: The biological characteristics of U937 / ADR cell line were significantly different from that of the parental U937 cell line. The multidrug resistance of multidrug-resistant U937 / ADR cell line was high, and the multidrug resistance protein P-gp was overexpressed. At the same time, the activation of p-Akt and NF -Kb.