摘要
目的 探讨与张力蛋白同源的10号染色体缺失的磷酸酶基因（phosphatase and tensin hemology deleted on chromosome ten gene,PTEN）及环氧化酶-2（cyclooxygenase-2,COX-2）在髓系白血病细胞中的表达及相互作用.方法 收集2007至2009年保定市第一医院及河北医科大学第二医院住院及门诊患者30例,其中10例慢性粒细胞白血病慢性期（chronic myeloid leukemia-chronic phase,CML-CP）患者、10例慢性粒细胞白血病急变期（chronic myeloid leukemia-blastic crisis,CML-BC）患者及10名健康人,分析所有研究对象骨髓单个核细胞内PTEN、COX-2信使核糖核酸（messenger ribonucleic acid,mRNA）表达水平变化.将携带有野生型PTEN和绿色荧光蛋白的腺病毒（Ad-PTEN-GFP）及对照载体腺病毒（Ad-GFP）转染人慢性粒细胞白血病（chronic myeloid leukemia,CML）细胞系K562.流式细胞仪检测转染效率；四甲基偶氮唑蓝（3-[4,5-dimethyl-2-thiazolyl]-2,5-dip,MTT）法检测细胞增殖抑制率及黏附功能；荧光定量聚合酶链反应（polymerase chain reaction,PCR）检测PTEN、COX-2 mRNA水平变化,蛋白质印迹（western blot,WB）检测PTEN、p-Akt蛋白表达水平的变化,细胞化学染色检测COX-2蛋白表达变化.结果 CML-BC患者中PTEN mRNA表达水平（0.022 ±0.021）低于CML-CP（1.134±1.124）及健康对照组（1.059±0.595,F=7.16,P＜0.01）,而COX-2 mRNA在CML-BC（0.761 ±0.418）患者中均高于CML-CP（0.211 ±0.158）及健康对照组（0.165±0.152,F=12.58,P＜0.01）.以MOI=200转染K562细胞后,Ad-PTEN-GFP组K562细胞最大增殖抑制率为38.67％ ±4.30％,明显高于Ad-GFP组抑制率（5.34％±0.31％,t=13.39,P＜0.01）,转染3d后Ad-PTEN-GFP组K562细胞内COX-2 mRNA表达水平（0.013 ±0.001）均明显低于Ad-GPF组（0.199±0.018）及未转染组COX-2 mRNA（0.217 ±0.021,F=499.45,P＜0.01）.p-Akt及COX-2蛋白表达水平亦明显减低.结论 PTEN可能通过抑制COX-2表达从而抑制白血病细胞增殖、黏附功能.
Objective To investigate expression and regulatory mechanism of phosphatase and tensin hemology deleted on chromosome ten gene （PTEN） and cyclooxygenase-2 （COX-2） in human myeloid leukemia cells.Methods Thirty patients was collected from the First Hospital of Baoding and Second Affiliated Hospital of Hebei Medical University,including 10 chroni myeloid leukemiac （CML）patients in chronic phase （CML-CP）,10 CML patients in blast crises （CML-BC） and 10 normal controls.The recombinated adenovirus containing green fluorescent protein （GFP） and PTEN （ Ad-PTEN-GFP） or empty vector （Ad-GFP） was transfected into human CML K562 cells.The growth of K562 cells and cell adhesion ability was observed by 3-[4,5-dimethyl-2-thiazolyl]-2,5-dip （MTF） assay; PTEN and COX-2 messenger ribonucleic acid （mRNA） levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR （FQ-PCR）.PTEN and p-Akt protein levels were detected by western blot,and COX-2 protein were measured with cytochemical staining.Results The mRNA expression levels of PTEN in CML-BC patients （0.022 ±0.021 ） were lower than CML-CP patients （1.134 ± 1.124） and normal control （ 1.059 ±0.595）.The mRNA expression levels of COX-2 in CML-BC patients （0.761 ±0.418） were higher than CML-CP （0.211 ± 0.158） and normal control （0.165 ± 0.152）.The growth of K562 cells was suppressed markedly,and the maximum growth inhibition rate was 38.67% ± 4.30% after transfected with PTEN gene,which was higher than Ad-GFP group （5.34% ±0.31%,t =13.39,P 〈0.01 ）.COX-2 mRNA expression levels in Ad-PTEN-GFP（0.013 ± 0.001 ）were significantly lower than Ad-GFP group （0.199 ±0.018） and untransfected group （0.217 ± 0.021,F =499.45,P 〈 0.01 ）,and p-Akt as well as COX-2protein were also down-regulated after K562 cells transfected （ MOI =200） with wild type PTEN in three days.Conclusion PTEN may inhibit proliferation and adhesion ability of leukemia cell in myeloid leukemia via down-regulating COX-2 expression.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2012年第2期165-169,共5页
Chinese Journal of Laboratory Medicine
基金
基金项目：河北省自然基金资助项目（C2010000538）
保定市科技攻关计划项目（11ZF003）
