摘要
目的:探讨PTEN/Akt/NF-κB信号通路对阿霉素耐药慢性粒细胞白血病细胞系K562/ADM细胞耐药逆转的分子作用机制。方法:将携带有野生型PTEN基因及绿色荧光蛋白的腺病毒(Ad-PTEN-GFP)或空载体(Ad-GFP)腺病毒转染K562/ADM细胞,在转染3d内联合应用不同浓度阿霉素(ADM),观察PTEN基因对阿霉素的协同抑制作用,根据IC50计算药物逆转倍数。通过MTT检测细胞增殖,流式细胞术检测转染效率、细胞凋亡率,荧光定量PCR(FQ-PCR)检测PTEN、NF-κB、I-κB、P53基因水平变化,Western blot检测PTEN、Akt、p-Akt、P65蛋白质水平变化。结果:Ad-PTEN-GFP转染与阿霉素联合作用后,K562/ADM细胞对阿霉素的敏感性增加,细胞增殖明显受抑,凋亡率明显高于Ad-GFP与阿霉素联合作用组,耐药逆转倍数为3.8倍。与Ad-GFP组相比,Ad-PTEN-GFP转染K562/ADM细胞3d后PTEN蛋白表达明显增加,p-Akt与P65蛋白表达明显下调,NF-κB mRNA表达水平下调,I-κB mRNA无明显改变,P53 mRNA表达轻度升高、Caspase-3/7活性增强。PTEN mRNA表达水平与NF-κB mRNA表达水平负相关(r=-0.968,P<0.05),NF-κB蛋白活性与Caspase-3/7活性负相关(r=-0.986,P<0.05)。结论:野生型PTEN可能通过抑制PI3K/NF-κB/Caspase信号传导通路,逆转K562/ADM细胞的多药耐药。
Objective：To investigate the mechanism of PTEN/Akt/NF-κB signal pathway in drug resistance reversal on K562/ADM cells in vitro. Methods： The recombinated adenovirus containing green fluorescent protein and PTEN（Ad-PTEN-GFP）or empty vector （Ad-GFP） was transfected into K562/ADM cells together with different concentration of adriamycin（ADM）. We detected the different drug IC50 with or without PTEN gene transfection to calculate the durg resistant factor （RF） by MTT; the proliferation of K562/ADM cells was observed by MTT assay; the apoptosis rate was assessed by flow cytometry （FCM）. PTEN, NF-κB, I-κB, P53 mRNA were detected by real-time fluorescent relative-quantification reverse transcriptional PCR （FQ-PCR）. PTEN, Akt, p-Akt and NF-κB protein levels were detected by western blot. Results： PTEN gene can enhance adriamycin sensitive in K562/ADM cells. The RF of ADM was 3.8 fold after transfection with PTEN gene. p-Akt and NF-Κb （P65） protein levels were down-regulated and NF-κB mRNA down-regulated too, but P53 up-regulated after transfaction with Ad-PTEN-GFP in K562/ADM cells, Caspase-3/7 activities up-regulated. PTEN mRNA expression levels were negative correlation with NF-κB mRNA（r=－0.968,P〈0.05）, NF-κB activities were negative correlation with Caspase-3/7 activities（r=－0.986,P〈0.05）. Conclusion： Wild PTEN gene might reverse adriamycin resistance via inhibiting PTEN/NF-κB/Caspase pathway.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2013年第3期54-60,共7页
China Biotechnology
