沉默核干因子基因表达对HL-60白血病细胞凋亡的影响  

Effects of Silent Nucleostemin Gene Expression on Apoptosis of HL-60 Cells In Vitro

作　　者：岳保红[1] 蔚利纳[1] 王园园[1] 

机构地区：[1]郑州大学第一附属医院检验科,郑州大学医学检验系,河南郑州450052

出　　处：《中国实验血液学杂志》2009年第2期319-323,共5页Journal of Experimental Hematology

基　　金：河南省医学创新人才工程基金资助项目(编号2005034);河南省郑州市科技攻关资助项目(编号074SGYS33196-14)

摘　　要：本研究探讨靶向沉默白血病细胞的核干因子(nucleostemin,NS)基因对HL-60白血病细胞凋亡的影响。用T7启动子介导体外合成两条短发夹状干扰RNA(NS-shRNA),以其中沉默NS基因作用强的一条NS-shRNA转染HL-60细胞,同时以与NS基因序列无关小干扰RNA转染HL-60细胞作对照,应用RT-PCR检测被转染的HL-60细胞NS-mRNA抑制效果,倒置显微镜下观察活体细胞形态变化,Wright-Giemsa染色观察细胞胞体和细胞核形态学变化,以流式细胞仪(FCM)和TdT介导的dUTP缺口末端标记技术(TUNEL)测定凋亡细胞数并计算凋亡阳性率。结果表明:HL-60细胞被NS-shRNA转染48小时后NS-mRNA阻断率达到74.94%,凋亡率分别为(25.32±3.06)%和(27.3±3.21)%,对照组仅(3.12±0.38)%和(3.30±1.52)%,转染组与对照组比较差异显著(p<0.01)。Wright-Giemsa染色显示细胞发生核碎裂并出现"凋亡小体"。结论:靶向沉默NS基因表达可诱发和促进HL-60白血病细胞发生凋亡,为NS基因作为恶性肿瘤治疗的候选靶基因奠定了理论基础。This study was aimed to explore whether the apoptosis of leukemia cells can be induced by targetingly silencing nucleostemin gene in vitro. HL-60 cells were taken as the model, and were directly transfected with nucleostemin short hairpin RNA （NS-shRNA）. Sequences unrelated with NS gene were taken as control. The blocking effect of NS-shRNA was detected by RT-PCR, the morphology changes in living cells were observed under inverted microscope, and the changes of cell shape and nucleus were detected by Wright-Giemsa staining. The amount of apoptotic cells were assayed by flow cytometer （FCM） and TUNEL technique, and the positive rate of apoptosis was determined meanwhile. The results showed that two NS-shRNA were synthesized in vitro, and the more effective one was selected to be transfected into HL-60 cells. The blocking rate of NS-mRNA reached to 74.94%. After transfection for 48 hours, Wright-Giemsa staining showed nuclear fragmentations and ＂apoptosis body＂ in cells. The apoptosis rate in transfected group detected by flow cytometry and TUNEL method were （ 25.32±3.06 ） % and （ 27.3 ± 3.21 ） % respectively, but were only （3. 12 ± 0. 38 ） % and （ 3. 30± 1.52 ） % in control group, the difference between the transfected group and the control group was significant （p 〈 0.01 ）. It is concluded that the apoptosis of HL450 leukemia cells can be induced by the silencing NS gene expression in vitro, which provides a theoretical basis for using NS gene as a candidate target gene in therapy of malignant tumor.

关 键 词：核干因子 细胞凋亡 短发夹状干扰RNA 白血病 HL-60细胞 

分 类 号：R733.7[医药卫生—肿瘤]