CAJ | 학술논문

目的探讨肿瘤抑制基因PTEN对人慢性粒细胞白血病(CML)中生存素(Survivin)、X连锁凋亡抑制蛋白(Xiap)、线粒体促凋亡蛋白(Smac)调控的作用.方法(1)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细胞系K562,四甲基偶氮唑盐(MTT)检测细胞增殖抑制率；流式细胞仪检测转染效率、细胞周期及凋亡率；荧光定量PCR(FQ-PCR)检测PTEN、Survivin、Xiap、Smac mRNA水平变化,Western印迹检测PTEN蛋白表达水平的变化.(2)研究10例慢性粒细胞白血病慢性期(CML-CP)、10例慢性粒细胞白血病急变期(CML-BC)及10名健康人(NC)骨髓单个核细胞内PTEN、Survivin、Xiap、Smac mRNA表达水平变化.结果以感染复数(MOI)=200转染K562细胞后,Ad-PTEN-GFP组K562细胞最大增殖抑制率为38.6％,转染3d后Ad-PTEN-GFP组K562细胞内Survivin、Xiap、Smac mRNA表达水平(0.0700±0.0059、0.0089±0.0006、0.0600±0.0039)均明显低于Ad-GPF组(0.4370±0.0790、0.0661±0.0072、0.1580±0.0078)和未转染组(0.4530±0.0810、0.0700±0.0079、0.1770±0.0085),转染Ad-PTEN-GFP组与转染Ad-GFP相比Survivin mRNA表达水平降低6.14倍、Xiap降低7.44倍,而Smac mRNA降低2.95倍(均P＜0.01).CML-BC患者中PTEN mRNA表达水平低于CML-CP及健康对照组,而Survivin、Xiap、Smac mRNA在CML-BC患者中均高于CML-CP及健康对照组(均P＜0.01).结论 Survivin、Xiap、Smac基因可能在PTEN介导的慢性粒细胞白血病细胞凋亡通路中参与抑制细胞增殖、促进细胞凋亡的作用.
목적 탐토종류억제기인PTEN대인만성립세포백혈병(CML)중생존소(Survivin)、X련쇄조망억제단백(Xiap)、선립체촉조망단백(Smac)조공적작용.방법(1)장휴대유야생형PTEN화록색형광단백적선병독(Ad-PTEN-GFP)급대조재체선병독(Ad-GFP)전염인만성립세포백혈병세포계K562,사갑기우담서염(MTT)검측세포증식억제솔；류식세포의검측전염효솔、세포주기급조망솔；형광정량PCR(FQ-PCR)검측PTEN、Survivin、Xiap、Smac mRNA수평변화,Western인적검측PTEN단백표체수평적변화.(2)연구10례만성립세포백혈병만성기(CML-CP)、10례만성립세포백혈병급변기(CML-BC)급10명건강인(NC)골수단개핵세포내PTEN、Survivin、Xiap、Smac mRNA표체수평변화.결과 이감염복수(MOI)=200전염K562세포후,Ad-PTEN-GFP조K562세포최대증식억제솔위38.6％,전염3d후Ad-PTEN-GFP조K562세포내Survivin、Xiap、Smac mRNA표체수평(0.0700±0.0059、0.0089±0.0006、0.0600±0.0039)균명현저우Ad-GPF조(0.4370±0.0790、0.0661±0.0072、0.1580±0.0078)화미전염조(0.4530±0.0810、0.0700±0.0079、0.1770±0.0085),전염Ad-PTEN-GFP조여전염Ad-GFP상비Survivin mRNA표체수평강저6.14배、Xiap강저7.44배,이Smac mRNA강저2.95배(균P＜0.01).CML-BC환자중PTEN mRNA표체수평저우CML-CP급건강대조조,이Survivin、Xiap、Smac mRNA재CML-BC환자중균고우CML-CP급건강대조조(균P＜0.01).결론 Survivin、Xiap、Smac기인가능재PTEN개도적만성립세포백혈병세포조망통로중삼여억제세포증식、촉진세포조망적작용.
Objective To explore the effects of tumor-suppressing gene wild type PTEN on the cell proliferation,apoptosis and the possible regulations of apoptosis-related molecules Survivin,Xiap and Smac gene in human chronic myeloid leukemia(CML)and cell line K562 cells.Methods(1)The recombinant adenovirus containing green fluorescent protein(GFP)and PTEN(Ad-PTEN-GFP)or empty vector(AdGFP)was transfected into K562 cells.The growth of K562 cells was observed by MTT assay while cell cycle and apoptotic rate were assessed by flow cytometry(FCM).PTEN,Survivin,Xiap and Smac mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR(FQ-PCR)while PTEN protein levels analyzed by Western blot.(2)The expression levels of PTEN,Survivin,Xiap and Smac mRNA were detected in 10 chronic myelogenous leukemia(CML)patients in chronic phase(CML-CP),10 CML patients in blast crises(CML-BC)and 10 normal control marrow mononuclear cells(MMNC).Results The growth of K562 cells was suppressed markedly.And the maximal growth inhibition rate was 38.6％ after the tranfection of PTEN.Survivin,Xiap,Smac mRNA expression levels were down-regulated by around 6.14,7.44 and 2.95 folds respectively(0.0700 ±0.0059,0.0089 ±0.0006,0.0600 ±0.0039 vs 0.4370 ± 0.0790,0.0661 ± 0.0072,0.1580 ± 0.0078 vs 0.4530 ± 0.0810,0.0700 ± 0.0079.0.1770 ±0.0085,all P ＜ 0.01).The mRNA expression level of PTEN in CML-BC patients was lower than that in CML-CP patients and normal control.But Survivin,Xiap,Smae mRNA expression levels were higher in CML-BC patients than those in CML-CP and normal control.Conclusion The over-expression of PTEN gene may inhibit the proliferation of K562 cells and promote cell apoptosis via the regulation of Survivin,Xiap and Smac genes.