沉默核干细胞因子基因对HL-60白血病细胞形态学和细胞化学影响的检测  

Detection of effects of nucleostemin gene silencing on morphology and cytochemistry of HL-60 cells in vitro

作　　者：岳保红[1] 佘红纯[2] 王清霞[1] 刘帅[1] 王园园[1] 蔚利纳[1] 张功员[3] 张钦宪[3] 

机构地区：[1]郑州大学第一附属医院检验科,河南郑州450052 [2]郑州铁路职业技术学院,河南郑州450052 [3]郑州大学基础医学院组织胚胎学教研室,河南郑州450052

出　　处：《检验医学》2009年第4期281-285,共5页Laboratory Medicine

基　　金：河南省医学创新人才工程基金资助项目(No.2005034);河南省郑州市科技攻关资助项目(No.074SGYS33196-14)

摘　　要：目的探讨体外沉默白血病细胞核干细胞因子(NS)基因转染后细胞形态学和细胞化学特征改变与细胞生物学行为的关系。方法体外合成NS特异性短发夹状RNA(shRNA)转染HL-60白血病细胞,逆转录-聚合酶链反应(RT-PCR)和免疫印迹法评价NS基因沉默和NS蛋白合成抑制效果,倒置显微镜观察活体细胞形态,Wright-Giemsa染色观察细胞形态学改变,血液分析仪测定细胞大小、粒度和均一性变化,髓过氧化物酶(MPO)、α-乙酸萘酯酶(α-NAE)和过碘酸-雪夫反应(PAS)测定反映细胞化学特征变化。结果沉默NS基因导致HL-60白血病细胞发生了形态学变化,细胞核和细胞质出现了进一步分化和成熟特征,Wright-Giemsa染色观察到细胞核碎裂现象,血液分析仪发现细胞变得大小不均并有较多核碎裂和无核的细胞碎片存在,细胞内定MPO、α-NAE酶活性和PAS反应增强。结论沉默NS基因表达能促使HL-60细胞进一步分化、成熟,细胞形态学和细胞化学特征发生相应改变,这些形态学变化可以作为研究细胞生物学行为改变的依据之一。Objective To explore the effect of nucleostemin （NS） gene silencing on morphology and cytochemistry of HL-60 cells, so to study the relationship between the changes of morphology and cytochemistry and biological behaviors. Methods HL-60 cells were directly transfected by NS specific short hairpin RNA （ shRNA ）. The effects of NS gene silencing and inhibition of NS protein synthesizing were detected by reverse transcription ploymerase chain reaction （RT-PCR） and Western blot. The morphology changes of HL-60 cells were observed by inverted microscope and Wright-Giemsa staining. The volume and granularity and size variation of HL-60 cells were assayed by blood cell analy- zer. Myeloperoxidase（MPO）, α-naphthol acetate esterase （α-NAE）and periodic acid-schiff （PAS） staining of HL-60 cells were analysed. Results The characters of differentiation and maturation in karyon and cytoplasm were observed by silencing NS gene in vitro. The nuclear fragmentations（karyorrhexises） showed in cells by Wright-Giemsa staining. The size variation of cells increased. The cells of small volume and karyorrhexises and cell-debrises without nucleus were found by blood cell analyzer. MPO and α-NAE activity and PAS positive rate increased after NS-shRNA transfection. Conclusions The redifferentiation and maturation of HL-60 cells can be induced by silencing NS gene in vitro. Mean- time, the characters of morphology and cytoehemistry of HL-60 cells would be altered according to differentiation, and these changes of morphology and cytochemistry may be one of evidences which can show biological behaviors of cells.

关 键 词：核干细胞因子 白血病 细胞形态学 细胞化学 HL-60细胞 

分 类 号：R733.7[医药卫生—肿瘤]