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PTEN基因逆转白血病多因素多药耐药机制探讨
成志勇1*,李琳1,王亚丽1,李华1,颜晓燕1,蒋丽媛1,田赫1,贾志强1,潘崚2
0
(1.保定市第一医院血液内科,保定 071000
2.四川大学华西医院血液内科, 成都 610041
*通信作者)
摘要:
目的 研究分析野生型PTEN基因对多柔比星(阿霉素,ADM)耐药人红白血病细胞系K562/ADM多药耐药(MDR)逆转的作用机制。方法 将携带野生型PTEN基因的腺病毒载体(Ad-PTEN-GFP)或空载体(Ad-GFP)感染ADM耐药的K562/ADM细胞,流式细胞术检测感染效率,在感染3 d内联合应用不同浓度的ADM、阿糖胞苷(Ara-C)或三氧化二砷(As2O3),通过MTT法检测细胞增殖,流式细胞术检测细胞凋亡率,根据IC50计算药物逆转倍数,观察PTEN基因对上述化疗药物MDR逆转作用。同时采用荧光定量PCR检测PTENNF-κBMDR1、MDR相关蛋白(MRP)及凋亡相关基因Bcl-2Bcl-xLBax水平变化,蛋白质印迹检测PTEN、Akt、p-Akt、P65水平变化。结果 以感染复数为200感染第3天后,Ad-PTEN-GFP感染与化疗药物联合作用组K562/ADM细胞增殖抑制率、凋亡率均高于Ad-GFP与化疗药物联合作用组(P<0.05),PTEN感染能增加K562/ADM对ADM、Ara-C、As2O3的敏感性,逆转倍数分别为3.80、2.65、2.64。与Ad-GFP组相比,Ad-PTEN-GFP 感染K562/ADM细胞3 d后p-Akt与P65蛋白表达下调,NF-κBMDR1Bcl-2Bcl-xL mRNA表达下调,Bax mRNA表达上调。结论 野生型PTEN基因可能通过抑制Akt信号转导通路进一步调控下游信号分子,通过下调NF-κB、MDR1、Bcl-2及上调Bax等多种信号分子逆转K562/ADM细胞的多药耐药。
关键词:  白血病  肿瘤抗药性  多药耐药  PTEN磷酸水解酶  Akt
DOI:10.3724/SP.J.1008.2013.00142
投稿时间:2012-10-18修订日期:2012-12-27
基金项目:保定市科技攻关计划(10ZF015).
Role of PTEN gene in multidrug resistance reversal in leukemia cells
CHENG Zhi-yong1*,LI Lin1,WANG Ya-li1,LI Hua1,YAN Xiao-yan1,JIANG Li-yuan1,TIAN He1,JIA Zhi-qiang1,PAN Ling2
(1. Department of Hematology, the First Hospital of Baoding, Baoding 071000, Hebei, China
2. Department of Hematology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China
*Corresponding author.)
Abstract:
Objective To investigate the mechanism by which wild-type PTEN gene reversing multi-drug resistance (MDR) in human leukemia K562/ADM cells resistant to adriamycin (ADM). Methods The recombinant adenovirus containing green fluorescent protein and PTEN (Ad-PTEN-GFP)or empty vector (Ad-GFP) was transducted into K562/ADM cells resistant to ADM. The transduction efficiency was assessed by flow cytometry (FCM). Then the cells were treated with different concentrations of ADM, cytarabine (Ara-C) or arsenic trioxide(As2O3) 3 days after transduction. The proliferation of K562/ADM cells was examined by MTT assay, the apoptosis rate was assessed by FCM, and the IC50 of different drugs was used to calculate the drug resistance reversal fold (RF), so as to observe the effect of PTEN on reversing MDR of the 3 drugs. PTEN, NF-κB, MDR1, MDR-associated protein (MRP) and apoptosis related genes (Bcl-2, Bcl-xL, Bax) were detected by fluorescence quantitative PCR. PTEN, Akt, p-Akt and NF-κB protein levels were detected by Western blotting analysis. Results The proliferation inhibition rate and apoptosis rate of cells in Ad-PTEN-GFP plus chemotherapeutic groups were significantly higher than those Ad-GFP plus chemotherapeutic groups at 3 days after infection (MOI=200) (P<0.05). PTEN transduction promoted the sensitivity of K562/ADM cells to ADM, Ara-C and As2O3, with the RF being 3.80, 2.65 and 2.64 folds, respectively. K562/ADM cells in Ad-PTEN-GFP group had lower p-Akt and NF-κB (P65) protein levels and lower NF-κB, MDR1, Bcl-2 and Bcl-xL mRNA levels, and up-regulated Bax mRNA level compared with those in Ad-GFP group. Conclusion Wild-type PTEN gene may reverse drug resistance via inhibiting Akt pathway and regulating its downstream signaling molecules, such as NF-κB, MDR1, Bcl-2 and Bax.
Key words:  leukemia  neoplasm drug resistance  multidrug resistance  PTEN phosphohydrolase  Akt