摘要
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 human multi-drug resistant leukemia cell line U937/ADR and investigate its biological characterization. Methods： Adriamycin resistant leukemia cell line U937/ADR was produced by high dose and short term methods; The cell growth and resistant index was tested by MTI＇; The cell cycle was detected by flow cytometry （FCM） ; Drug excretion was tested by rhodamine123 （Rho123） ; MDR1, MRP1, NF-Kb, Bcl-2, Bax mRNA were detected by real-time fluorescent relative-quantification reverse transcriptional PCR （FQ-PCR） ; P-gp, MRP1, Akt, p-Akt and p65 protein levels were detected by Western blot. Results： Drug resistant cell line U937/ADR was successfully established and the ADR resistant index was 11 fold than U937 cells. The doubling time of U937/ADR cells were 43.6h and longer than that of U937 cells. Cell cycle showed that the GO/G1 phase cells increased and G2/M phase cells decreased in U937/ADR cells and it showed the cross resistant to many drugs and drug excretion increased. MDR1, NF-Kb, Bcl-2 mRNA and P- gp, P65, Bcl-2 and p-Akt protein increased in U937/ADR cells. Conclusion： The biological characterizations of U937/ADR were different to those of U937, which showed the cross resistant to many drugs and over-expression of the P-gp, activated NF-Kb and p-Akt.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2009年第7期17-21,共5页
China Biotechnology
基金
河北省科技攻关计划资助项目(072761130)
关键词
U937
多药耐药
阿霉素
P糖蛋白
转录因子
U937 cell line Multidrug resistance Adriamycin P-glucoprotein Transcription factor
