PTEN基因逆转白血病多因素多药耐药机制探讨

Role of PTEN gene in multidrug resistance reversal in leukemia cells

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PTEN、NF-κB、MDR1、MDR相关蛋白(MRP)及凋亡相关基因Bcl-2、Bcl-xL及Bax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-κB、MDR1、Bcl-2、Bcl-xLmRNA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。

Objective To investigate the mechanism by which wild-type PTEN gene reversing multi-drug resistance （MDR） in human leukemia K562/ADM ceils 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 eytometry （FCM）. Then the cells were treated with different concentrations of ADM, cytarabine （Ara-C） or arsenic trioxide（As2 03 ） 3 days after transduction. The proliferation of K562/ADM cells was examined by MTT assay, the apoptosis rate was assessed by FCM, and the ICs0 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-B, MDR1, MDR-associated protein （MRP） and apoptosis related genes （Bcl-2, Bcl-：rL, Bax） were detected by fluorescence quantitative PCR. PTEN, Akt, p-Akt and NF-B 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 （MOI200） （P〈0.05）. PTEN transduction promoted the sensitivity of K562/ADM cells to ADM, Ara-C and As203, 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-B （P65） protein levels and lower NF-：B, MDR1, Bcl-2 and Bcl-：rL 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 downstreamsignaling molecules, such as NF B, MDR1, Bcl-2 and Bax.