shRNA干扰CIAPIN1基因表达促进K562细胞粒系分化  

CIAPIN1 gene silence induces granulocytic differentiation of K562 cells

作　　者：王建[1] 王迟鹃 许华[1] 张丽媛[2] 蔺亚妮[1] 常国强[1] 梁昊岳 茹永新[1] 李庆华[1] 张洪菊[1] 张海瑞[1] 庞天翔[1] 

机构地区：[1]中国医学科学院 北京协和医学院 血液学研究所血液病医院 实验血液学国家重点实验室,天津300020 [2]天津市第四中心医院急诊科

出　　处：《中华临床医师杂志（电子版）》2013年第20期77-80,共4页Chinese Journal of Clinicians(Electronic Edition)

基　　金：国家自然科学基金(81170510)

摘　　要：目的探讨靶向干扰CIAPIN1基因表达对慢性粒细胞白血病(CML)细胞系K562粒系分化的影响。方法针对CIAPIN1基因设计并构建短发卡RNA(shRNA)真核表达载体,转染K562细胞并作为干扰组,以无关序列shRNA处理的K562细胞作为对照组。采用Real-time PCR及Western blot技术鉴定干扰效率;采用瑞氏染色和电镜分别观测K562细胞形态学和超微结构的改变;采用Real-time PCR方法检测粒系分化标志基因中性粒细胞胞浆因子1(NCF1)、血清黏蛋白1(ORM1)以及粒系分化转录因子CCAAT/增强子结合蛋白(C/EBPα)、低氧诱导因子1α(HIF1α)mRNA水平的改变;流式细胞术检测K562细胞表面分化抗原CD11b表达的改变;Western blot技术检测ERK1/2、JNK、p38MAPK及Akt磷酸化水平的改变。结果与对照组相比,干扰组的CIAPIN1基因表达被有效抑制(P<0.05);K562细胞的形态和超微结构趋向成熟粒细胞阶段;NCF1、ORM1、C/EBPα及HIF1αmRNA表达水平升高(P<0.05);CD11b表达升高(P<0.01);ERK1/2磷酸化水平升高(P<0.05),而JNK、p38MAPK及Akt磷酸化水平未见明显改变。结论抑制CIAPIN1基因表达能促进K562细胞向成熟的粒细胞阶段分化,ERK1/2磷酸化水平的升高可能参与了该分化过程。Objective The study was aimed to investigate the effect of CIAPIN1 gene silence by shRNA interference on the granulocytic differentiation of chronic myeloid leukemia （CML） cell line K562. Methods The shRNA eukaryotic expression vector targeting CIAPIN1 gene was constructed and transfected into K562 cells. K562 cells were divided into interference group （K562 cells transfected with CIAPIN1 gene interfering plasmid DNA ） and control group （K562 cells lransfected with unrelated sequence plasmid DNA）. The inhibition efficiency was detected by Real-time PCR and Western blot; the morphological and ultramicrostructural changes were observed using inverted microscopy and transmission electron microscopy, respectively; the mRNA levels of Neutrophil Cytosolic Factor 1 （NCF1）, Orosomucoid 1 （ORMI）, CAAT/enhancer binding protein （C/EBPα） and Hypoxia Inducible Factor 1α （HIF1α） were detected by Real-time PCR; the granulocyte specific cell surface maturation marker CD1 lb expression was analyzed by flow cytometry （FCM）; the phosphorylated ERK1/2, JNK, p38MAPK and Akt expression were assessed by Western blot. Results Compared with control group, the CIAPIN1 gene expression was statistically decreased （P〈0.05）; the morphology and ultramicrostructure of K562 cells were consistent with that of mature granulocytes; the mRNA levels of NCF1, ORM1, C/EBPα and HIFlct were statistically increased （P〈0.05）; the expression levels of CDllb and phosphorylated ERK1/2 were significantly up-regulated （P〈0.05）. CIAPIN1 gene silence had no effect on the expression level changes of phosphorylated JNK, p38MAPK and Akt. Conclusion Inhibition of CIAPINI gene expression induced K562 cells mature granulocytic differentiation. The differentiation was possibly mediated by the up-regulation of phosphorylated ERK1/2.

关 键 词：K562细胞 CIAPIN1基因 SHRNA干扰 粒系分化 

分 类 号：R363[医药卫生—病理学]