CAJ | 학술논문

目的探讨上调抑癌基因p14ARF表达对慢性髓性白血病(CML)细胞凋亡的影响以及其对伊马替尼增敏的作用.方法应用水泡性口膜炎病毒外壳糖蛋白假构型(VSV-G pseudotyped)慢病毒载体基因转导系统,将抑癌基因p14ARF导入CML细胞系K562及4例CML急变期患者原代白血病细胞(分别称为CML-BC1 ～4),同时以转染空载体的细胞作为对照组.用荧光显微镜及流式细胞术检测转导效率,Western blot法检测K562细胞p14ARF蛋白表达；用WST-8法检测细胞增殖抑制率及转染目的基因的K562细胞在不同浓度倍比稀释的伊马替尼(0、0.015、0.062、0.125、0.25、0.5、1.0、2.0 μmol/L)处理后细胞增殖抑制情况；用FITC标记的膜联蛋白V/碘化丙锭(Annexin V-FITC/PI)标记流式细胞术检测细胞凋亡；半固体培养法检测白血病细胞集落形成能力.结果荧光显微镜及流式细胞术检测GFP阳性细胞率显示,转染目的基因及空载体的K562细胞及CML-BC1细胞转染效率接近100％,而CML-BC2 ～4细胞转导效率为80％～90％；Western blot分析结果显示,转导p14ARF的K562细胞(K562-p14ARF)ARF蛋白表达较未转导细胞明显提高；K562-p14ARF细胞凋亡率为20％；转导p14ARF后的4例CML患者原代白血病细胞凋亡率为(71.1±22.4)％,与对照组的(12.4±6.2)％相比差异有统计学意义(P＜0.05).伊马替尼对K562-p14ARF细胞增殖有明显的抑制作用,且呈浓度依赖性.转导p14ARF后的4例CML患者原代白血病细胞集落形成数为41.5 ±13.2,与对照组(88.5±7.9)相比差异有统计学意义(P＜0.05).结论人为上调p14ARF基因表达可诱导CML细胞凋亡,与伊马替尼联合应用可进一步加强对细胞增殖的抑制作用.
목적 탐토상조억암기인p14ARF표체대만성수성백혈병(CML)세포조망적영향이급기대이마체니증민적작용.방법 응용수포성구막염병독외각당단백가구형(VSV-G pseudotyped)만병독재체기인전도계통,장억암기인p14ARF도입CML세포계K562급4례CML급변기환자원대백혈병세포(분별칭위CML-BC1 ～4),동시이전염공재체적세포작위대조조.용형광현미경급류식세포술검측전도효솔,Western blot법검측K562세포p14ARF단백표체；용WST-8법검측세포증식억제솔급전염목적기인적K562세포재불동농도배비희석적이마체니(0、0.015、0.062、0.125、0.25、0.5、1.0、2.0 μmol/L)처리후세포증식억제정황；용FITC표기적막련단백V/전화병정(Annexin V-FITC/PI)표기류식세포술검측세포조망；반고체배양법검측백혈병세포집락형성능력.결과 형광현미경급류식세포술검측GFP양성세포솔현시,전염목적기인급공재체적K562세포급CML-BC1세포전염효솔접근100％,이CML-BC2 ～4세포전도효솔위80％～90％；Western blot분석결과현시,전도p14ARF적K562세포(K562-p14ARF)ARF단백표체교미전도세포명현제고；K562-p14ARF세포조망솔위20％；전도p14ARF후적4례CML환자원대백혈병세포조망솔위(71.1±22.4)％,여대조조적(12.4±6.2)％상비차이유통계학의의(P＜0.05).이마체니대K562-p14ARF세포증식유명현적억제작용,차정농도의뢰성.전도p14ARF후적4례CML환자원대백혈병세포집락형성수위41.5 ±13.2,여대조조(88.5±7.9)상비차이유통계학의의(P＜0.05).결론 인위상조p14ARF기인표체가유도CML세포조망,여이마체니연합응용가진일보가강대세포증식적억제작용.
Objective To investigate the effect of up-regulated expression of tumor suppressor gene p14ARF on apoptosis of chronic myeloid leukemia (CML) cells and its interaction with imatinib.Methods Tumor suppressor gene p14ARF was transduced into K562 (K562-p14ARF) and 4 blast crisis primary CML cells (CML-BC 1-4) using vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector with cells transduced by empty vector as control.Fluorescence microscopy and flow cytometry were applied to measure transduction efficiency,and Western blotting assay was used to detect p14ARF protein of K562 cells.WST-8 method was used to determine cell growth inhibition rate of K562 cells transduced by the target gene under different concentrations of imatinib (0,0.015,0.062,0.125,0.25,0.5,1.0,2.0 μmol/L).Cell apoptosis and leukemic cellular colony-forming ability were detected by Annexin V-FITC/PI dyeing using flow cytometry (FCM) and semi-solid culture method respectively.Results Fluorescence microscopy and FCM showed that transduction efficiency (GFP positive cells) of K562-p14ARF,K562-VSV and CML-BC1 cells were close to 100％,and CML-BC 2-4 cells were 80％ to 90％ on average.Results of Western blotting showed that the levels of ARF protein expression of K562 cells transduced by p14ARF were significantly higher than of untransduced cells; the apoptosis rate of K562-p14ARF was 20％ ; the mean apoptosis rate of 4 primary leukemic cells transduced by the p14ARF [(71.1 ± 22.4) ％] was significantly higher than of control group [(12.4 ± 6.2) ％] (P ＜ 0.05).Imatinib significantly inhibited the proliferation of K562-p14ARF cells in a dose-dependent manner.The mean leukemic cellular colony-forming unit of 4 primary leukemic cells transduced by the p14ARF(41.5 ± 13.2) was significantly lower than of the control group (88.5 ±7.9) (P ＜0.05).Conclusion Increased p14ARF gene expression could induce apoptosis of CML cells; Moreover,it could enhance inhibitory effect on cell proliferation when combined with imatinib.