摘要
目的探讨AML1/ETO融合基因沉默后kasumi-1细胞株对组蛋白去乙酰化酶抑制剂(HDACI)丙戊酸钠(VPA)的敏感性变化。方法体外培养人急性髓系白血病细胞株kasumi-1,脂质体介导重组质粒pGCsiRNA-AML1/ETO转染kasumi-1,实时荧光定量PCR(RT-PCR)检测Kasumi-1细胞AML1/ETO和Bcl-2 mRNA的表达变化;MTT法检测VPA对细胞增殖的影响;流式细胞术(FACS)检测细胞周期;Hochest33258染色后荧光显微镜下观察细胞凋亡的形态学变化。结果靶向AML1/ETO基因的siRNA质粒可有效降低Kasumi-1细胞AML1/ETO基因的表达;与空白对照组相比,转染组细胞AML1/ETO基因mRNA表达量下降了53.7%(P<0.05),同时Bcl-2 mRNA的表达量下降了54.5%(P<0.05),而阴性对照组及脂质体组两基因的表达量无明显变化;VPA对Kasumi-1细胞增殖的抑制作用呈浓度、时间依赖性,不同VPA浓度作用下,转染组细胞24、48 h的抑制率均高于空白对照组(P<0.05),而阴性对照组及脂质体组与空白对照组相比无明显变化。空白对照组及转染组细胞G0/G1期比例分别为(42.07±5.23)%和(62.6±5.87)%(P<0.01),经含2 mmol/L VPA的培养基培养48 h后,两组细胞G0/G1期比例分别上升至(69.2±7.02)%和(78.92±6.23)%(P<0.01);转染后细胞出现核固缩、核边集、凋亡小体等改变。结论特异性AML1/ETO siRNA可显著抑制Kasumi-1细胞AML1/ETO基因的表达,并明显增强Kasumi-1细胞对VPA的敏感性。
Objective To investigate the sensitivity of Kasumi-1 cells to sodium valproate（VPA）,a histone acetylation enzyme inhibitor（HDACI）,after silence of the AML1/ETO gene.Methods The plasmid pGCsiRNA-AML1/ETO was transfected into Kasumi-1 cells by liposomes.Expressions of AML1/ETO and Bcl-2 mRNA were assayed by real time PCR（RT-PCR）.The effect of VPA on proliferation of Kasumi-1 cells was assessed by MTT.The cell cycle was assessed by flow cytometry.The morphology of apoptosis after Hochest33258 staining was observed under a fluorescence microscope.Results After transfection with pGCsiRNA-AML1/ETO,expression of AML1/ETO mRNA in Kasumi-1 cells was reduced by 53.7% compared with the controls（P〈0.05）,while expression of Bcl-2 mRNA was reduced by 54.5%（P〈0.05）.The inhibitory effect of VPA on proliferation of Kasumi-1 cells was concentration-and time-dependent,and inhibitory rates in the transfected group were higher than those in the control group 24 h and 48 h after transfection,with different VPA concentrations.Rates of cells in the G0/G1 phase in the control group and the transfected group were（42.07±5.23）% and（62.6±5.87）%,respectively（P〈0.01）.After the treatment with VPA（2 mmol/L）for 48 h,the rates increased to（69.2±7.02）% and（78.92±6.23）%,respectively（P〈0.01）.After transfection,cells presented karyopyknosis,nuclear margination and apoptotic bodies.Conclusion siRNA specifically targeting the AML1/ETO gene can remarkably down-regulate expression of the AML1/ETO gene,and enhances the sensitivity of Kasumi-1 cells to HDACI.
出处
《山东大学学报（医学版）》
CAS
北大核心
2011年第7期68-73,共6页
Journal of Shandong University:Health Sciences
