摘要
目的 抑制SALL4基因在急性白血病细胞THP-1中的表达,探讨其在白血病发病机制中的作用.方法 将表达SALL4干扰序列的质粒载体转染至急性白血病细胞THP-1中,分为4组：（1）空白对照组：不加处理;（2）转染对照组：加入空pRS质粒载体;（3）转染一组：加入SALL4-shRNA-pRS-1干扰质粒的转染复合体;（4）转染二组：加入SALL4-shRNA-pRS-2干扰质粒的转染复合体.采用实时荧光定量PCR和WB技术观察THP-1细胞中SALL4基因mRNA及蛋白表达的变化,建立抑制SALL4表达的体系.实时荧光定量PCR检测SALL4受抑后Wnt/β-catenin信号通路中C-myc、Cyclin D1、β-catenin基因的表达改变,采用流式细胞术分析THP-1细胞凋亡情况.结果 实时荧光定量PCR结果 显示,转染一组、转染二组、转染对照组、空白对照组SALL4基因的表达水平分别为（36.0±4.3）%、（32.0±2.4）%、（102.0±6.5）%、（100.0±2.6）%,差异有统计学意义（F=226.3,P〈0.05）;转染一组、转染二组的SALL4基因表达水平显著低于空白对照组,差异有统计学意义（t值分别为19.7、19.1,P〈0.05）;转染对照组SALL4基因表达水平与空白对照组比较差异无统计学意义（t=1.1,P＞0.05）.WB检测结果 显示,转染一组、转染二组SALL4蛋白表达与空白对照组和转染对照组相比明显下降.以上基因和蛋白表达水平均提示SALL4干扰效率良好.SALL4基因抑制表达后,转染一组C-myc基因、β-catenin基因、Cyclin D1基因的表达分别为（44.0±6.2）%、（44.0±5.1）%和（107.0±13.6）%,转染二组为（22.0±4.5）%、（25.0±3.5）%和（48.0±7.6）%,转染对照组为（42.0±3.5）%、（59.0±3.7）%及（79.0±5.6）%.转染一组、转染二组C-myc基因表达水平显著低于空白对照组的（103.0±7.5）%,差异有统计学意义（t值分别为10.1、9.5,P均〈0.05）;转染一组、转染二组β-catenin基因的表达显著低于空白对照组的（100.0±4.1）%,差异有统计学意
Objective To inhibit the expression level of SALI4 in AML cell line THP-1 and investigate its potential effects on pathogenesis of leukemia. Methods AML cell line THP-1 was transfected with plasmids that expressed small interfering RNA targeting SALL4. The samples were divided into 4 groups：（1） blank group： samples with not any treatments; （2） control group： cells with empty pRS vector alone;（3） test1 group：cells with SALL4-shRNA-pRS-1 plasmid transfection complex; （4） test2 group：cells with SALL4-shRNA-pRS-2 plasmid transfection complex. The expression levels of SALL4 mRNA and protein were measured by real time fluorescence quantitative PCR and WB. C-myc, Cyclin D1 and β-catenin were important components of Wnt/β-catenin signaling pathway and their expression levels in SALL4 knockdown THP-1 cells were detected by real-time fluorescence PCR. Furthermore, THP-1 apoptosis was analyzed by flow cytometry after Annexin V-PI staining. Results Real time fluorescent quantitative PCR illustrated that the expression of SALL4 in testl group, test2 group, control group and blank group were （ 36. 0 ± 4. 3 ） %,（32. 0 ± 2. 4） %, （ 102. 0 ± 6.5 ） % and （ 100. 0 ± 2. 6 ） % respectively. There was statistical significance （ F = 226. 3, P 〈 0. 05 ）. The expression of SALL4 in testl and test2 group respectively were significant lower than that in blank group （t = 19.7,19. 1, P〈0. 05）. The expression of SALL4 had no significant difference between blank group and control group （t = 1.1, P ＞0. 05）. Western blot analysis revealed SALL4 protein in testl and test2 group were significantly decreased compared with those of control and blank group. All above data indicated the high efficiency of RNA interference targeting SALL4. Comparing with the blank group, the relative expression of C-myc, Cyclin D1 and β-catenin mRNA in test1, test2 and control group were（44.0 ±6.2）%,（44.0 ±5.1）% and （107.0±13.6）%;（22.0±4.5）%,（25.0±3.5）% and （48.0 ± 7. 6 ） %
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2010年第12期1202-1207,共6页
Chinese Journal of Laboratory Medicine
基金
国家自然科学基金资助项目（30770907）
关键词
白血病
细胞系
肿瘤
转录因子
RNA干扰
细胞凋亡
Leukemia
Cell line, tumor
Transcription factors
RNA interference
Apoptosis
