TBLR1-RARα融合基因对K562细胞向红系分化的影响  

Effect of TBLR1-RARα Fusion Gene on Erythroid Differentiation of K562 Cells

作　　者：陈晶[1] 李欢[1] 安娜[1] 李寿芸 卢文婷[1] 邢海燕[1] 饶青[1] 王敏[1] 王建祥[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实验学血液学国家重点实验室,天津300020

出　　处：《中国实验血液学杂志》2015年第6期1702-1708,共7页Journal of Experimental Hematology

基　　金：国家自然基金项目编号(81370633;81300380);天津市应用基础研居重点项目(13JCZDJC2990)

摘　　要：目的:探讨融合基因TBLR1-RARα的表达对于白血病细胞系K562细胞向红系分化的作用及其可能的机制。方法:应用Tet-Off系统,通过强力霉素(doxycycline,Dox)调控TBLR1-RARα的条件性表达。将TBLR1-RARα融合基因与慢病毒目的载体p LVX-Tight-Puro连接,感染K562细胞并获得稳定克隆,Western blot证实TBLR1-RARα融合蛋白的表达情况。应用实时定量荧光PCR检测全反式维甲酸(all-trans retinoid acid,ATRA)处理的K562细胞红系分化的表面标志CD71以及α,ε,γ-珠蛋白的基因表达水平;流式细胞术(flow cytometry)分析细胞表面CD71和CD235a的表达情况,联苯胺染色观察血红蛋白的生成情况。结果:实时定量PCR显示,ATRA能够使这些细胞的红系分化相关的CD71及α,ε,γ-珠蛋白的基因表达水平上调;流式分析结果同样表明,ATRA促使红系分化早期标志CD71细胞所占比例增加。ATRA处理后,联苯胺染色证实这些细胞的胞浆出现蓝染,表明ATRA能够诱导表达TBLR1-RARα的K562细胞产生血红蛋白。结论:TBLR1-RARα融合基因表达,有利于ATRA诱导K562细胞向红系分化。Objective:To explore the effects of TBLR1-RARα on the differentiation induction of leukemia cell line K562 cells into erythroid lineage and to investigate its related mechanisms.Methods:Tet-Off inducible system was used to construct the conditional expression vector of TBLR1-RARα fusion gene by cloning the TBLRl-RARa fragment into lentivirus vector pLVX Tight-Puro,the expression of TBLRl-RARα fusion gene was induced by doxycycline(Dox).Then,K562 cells were transfected with lentivirus pLVX Tight-Puro-TBLRl-RARa-flag,and the expression of fusion proteins was verified by Western blot.After treatment of K562 with all-trans retinoid acid(ATRA),real time RT-PCR was performed to test the expression of erythroid differentiation-related CD71 and α,ε,γ-globins gene.Flow cytometry was used also to analyze the expression of erythroid differentiation markers CD71 and CD235 a.Benzidine staining was used to detect the production of hemoglobin in K562 cells.Results:qRT-RCR showed that ATRA could increase the expression level of CD71 and α,ε,γ-globin genes when TBLR1-RARa was expressed.After treatment of ATRA,the proportion of CD71^+ cells detected by the flow cytometry also increased.Benzidine staining showed that ATRA could induce hemoglobin production in K562 cells with TBLRl-RARa fusion gene expression.Conclusion:The expression of TBLRl-RARα fusion gene contribute to ATRA inducing differentiation of K562 cells into erythroid lineage.

关 键 词：TBLR1-RARα融合基因 K562 全反式维甲酸 红系分化 

分 类 号：R733.71[医药卫生—肿瘤]