CAJ | 학술논문

目的探讨CCAAT增强子结合蛋白α(C/EBPα)在体内和体外对急性粒细胞白血病HL60细胞分化和凋亡的影响及其可能机制.方法将C/EBPα表达质粒pEGFP-C/EBPα经阳离子脂质体介导转染HL60细胞,用G418筛选出C/EBPα稳定表达细胞株为转染组;以转染pEGFP空载质粒HL60细胞为空载体组;未处理的HL60细胞为对照组.瑞氏染色观察细胞形态学变化;噻唑蓝(MTT)比色法检测细胞增殖抑制率;流式细胞术分析凋亡;RT-PCR及免疫印迹法(Western blot)检测c-myc基因的表达.20只BALB/c裸鼠按完全随机法分为3组:转染组7只、空载体组7只、对照组6只.将3组细胞分别皮下注射相应各组小鼠形成皮下瘤,20 d后处死小鼠,测量肿瘤质量及大小,TUNEL法检测皮下瘤组织细胞凋亡率.结果筛选到C/EBPα基因稳定表达的细胞株pEGFP-C/EBPα-HL60,细胞出现明显分化.对照组、空载体组和转染组细胞的凋亡率分别为(5.4±1.4)%、(5.0±1.3)%、(21.9±4.5)%,转染组细胞的凋亡率显著高于对照组和空载体组(均P＜0.05).RT-PCR及免疫印迹显示C/EBPα明显抑制c-myc mRNA和蛋白的表达(均P＜0.05).对照组、空载体组和转染组细胞接种小鼠形成皮下瘤,其质量分别为(5.35±1.12)g、(5.12±1.31)g和(3.26±0.72)g,瘤体大小分别为(25±4)mm、(18±3)mm和(11±2)mm,转染组瘤体质量及瘤体大小明显低于对照组和空载体组(均P＜0.05).与对照组和空载体组比较,转染组皮下瘤组织细胞凋亡率明显增加(均P＜0.05).结论 C/EBPα在HL60细胞中具有促进细胞凋亡和抑制细胞增殖的能力,显示C/EBPα在急性粒细胞白血病中是一种肿瘤抑制因子.
목적 탐토CCAAT증강자결합단백α(C/EBPα)재체내화체외대급성립세포백혈병HL60세포분화화조망적영향급기가능궤제.방법 장C/EBPα표체질립pEGFP-C/EBPα경양리자지질체개도전염HL60세포,용G418사선출C/EBPα은정표체세포주위전염조;이전염pEGFP공재질립HL60세포위공재체조;미처리적HL60세포위대조조.서씨염색관찰세포형태학변화;새서람(MTT)비색법검측세포증식억제솔;류식세포술분석조망;RT-PCR급면역인적법(Western blot)검측c-myc기인적표체.20지BALB/c라서안완전수궤법분위3조:전염조7지、공재체조7지、대조조6지.장3조세포분별피하주사상응각조소서형성피하류,20 d후처사소서,측량종류질량급대소,TUNEL법검측피하류조직세포조망솔.결과 사선도C/EBPα기인은정표체적세포주pEGFP-C/EBPα-HL60,세포출현명현분화.대조조、공재체조화전염조세포적조망솔분별위(5.4±1.4)%、(5.0±1.3)%、(21.9±4.5)%,전염조세포적조망솔현저고우대조조화공재체조(균P＜0.05).RT-PCR급면역인적현시C/EBPα명현억제c-myc mRNA화단백적표체(균P＜0.05).대조조、공재체조화전염조세포접충소서형성피하류,기질량분별위(5.35±1.12)g、(5.12±1.31)g화(3.26±0.72)g,류체대소분별위(25±4)mm、(18±3)mm화(11±2)mm,전염조류체질량급류체대소명현저우대조조화공재체조(균P＜0.05).여대조조화공재체조비교,전염조피하류조직세포조망솔명현증가(균P＜0.05).결론 C/EBPα재HL60세포중구유촉진세포조망화억제세포증식적능력,현시C/EBPα재급성립세포백혈병중시일충종류억제인자.
Objective To investigate the effect of CCAAT enhancer binding protein α(C/EBPα) on differentiation and apoptosis in the acute myeloid leukemia HL60 cells in vitro and in vivo and its possible mechanism. Methods The C/EBPα expression plasmid pEGFP-C/EBPα and empty control plasmid were respectively transfected into HL60 cells with cationic liposome as transfected group and empty plasmid transfected group, and untreated HL60 cells served as control group. The cells stably expressing the C/EBPα gene were obtained by G418 selection. The morphological changes were observed under light microscope following WrightGiemsa staining. MTT assay was employed to evaluate cell proliferation, and flow cytometry(FCM) was performed to analyze cell apoptosis. Meanwhile, the expression of c-myc was respectively detected by RT-PCR and Western blot both at the mRNA and protein level. Twenty BALB/c nude mice were divided into 3 groups in a completely randomized design: 7 mice in transfected group, 7 mice in empty plasmid transfected group and 6 in control group. Three kinds of cells including pEGFP-C/EBPα-HL60 cells, pEGFP -HL60 cells and the control HL60 cells were injected into mice separately through the subcutaneous. The mice were sacrificed at 20 d after injection. The mass and size of subcutaneous xenograft tumors were measured and the cell apoptosis of subcutaneous tumor were detected by TUNEL. Results The pEGFP-C/EBPα-HL60 cell line stably expressing the C/EBPα gene was screened out. Compared to either empty plasmid transfected group or control group, the expression of C/EBPα could promote cellular differentiation of HL60. FCM showed higher apoptotic rate in transfected group[ (21.9±4.5)%,P＜0.05 ] ,while (5.4±1.4)% in control group and (5.0±1.3)% in empty plasmid transfected group. c-myc expression was significantly down-regulated by C/EBPα both at the mRNA and protein level. The mass and size of tumors in transfected group were smaller than those in empty plasmid transfected group and control group [ (5.35±1.12)g and(25±4)mm in control group, (5.12±1.31)g and ( 18±3)mm in empty plasmid transfected group ,while (3.26±0.72)g and ( 11±2)mm in transfected group, all P＜0.05]. More apoptosis cells were found in subcutaneous tumor of transfected group(both P＜0.05). Conclusion C/EBPα can not only inhibit the proliferation, but also induce massive apoptosis of HL60 cells, meanwhile C/EBPα is a tumor suppressor of acute myeloid leukemia.