bcr-abl融合基因特异性siRNA对K562细胞生物学特性的影响  

Effect of specific siRNA targeting against bcr-abl chimeric gene on chronic myelogenous leukemia cells

作　　者：王莎[1] 柴玉波[2] 刘飞[3] 张孝勇[4] 贾卫[3] 谢鑫[3] 于文强[1] 尚振川[1] 金伯泉[3] 孙秉中[1] 

机构地区：[1]第四军医大学西京医院血液内科,西安710032 [2]第四军医大学基础部化学与分子生物学教研室,西安710032 [3]第四军医大学基础部免疫学教研室,西安710032 [4]第四军医大学唐都医院放射科

出　　处：《中华医学杂志》2005年第3期198-202,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目(39770336;30170361)

摘　　要：目的应用特异性siRNA(小干涉RNA)抑制慢性粒细胞白血病bcrabl融合基因表达,观察其对K562细胞生物学特性的影响。方法以慢性粒细胞白血病细胞系K562为研究对象,合成并转染针对K562细胞bcrabl融合基因融合位点的21ntsiRNA,应用Westernblot法检测bcrabl融合基因的表达;3HTdR掺入法检测K562细胞的增殖活性;AnnexinⅤFITC/PI染色法检测K562细胞的存活状况;流式细胞仪法检测K562细胞周期变化以及联苯胺染色法检测K562细胞的分化状况;应用Westernblot法检测凋亡相关蛋白BclxL/Bax的表达。结果(1)RNA干涉组K562细胞bcrabl融合基因的表达水平明显下降;(2)RNA干涉组的CPM值(每分钟脉冲数)在siRNA转染K562细胞第24h,48h,72h和96h均明显低于对照组(下降率依次为3306%,5225%,5764%,7087%),(3)RNA干涉组转染48h时有432%的K562细胞发生凋亡;(4)RNA干涉组K562细胞凋亡相关蛋白BclxL的表达水平下调,但Bax的表达无明显变化;(5)RNA干涉组K562细胞联苯胺染色阳性比例增加,部分K562细胞向红系分化;(6)RNA干涉组K562细胞出现明显的G1期阻滞。结论特异性siRNA分子可以显著抑制bcrabl融合基因的表达,影响K562细胞的基本生物学特性,最终导致K562细胞分化或凋亡。Objective To investigate the effect of specific small interfering RNA (siRNA) targeting against bcr-abl chimeric gene on the biological traits of chronic myelogenous leukemia (CML) cells. Methods CML cells of the line K561 transcribing a type of b3: a2 mRNA of bcr-abl chimeric gene were cultured. A 21nt siRNA targeting against the chimeric location of the b3: a2 mRNA of bcr-abl chimeric gene was designed, synthesized, and transfected into the K562 cells as RNA interference group. Another K562 cells were transfected with fluorescein enzyme gene specific siRNA as indifferent controls, or with lipid alone as blank vector controls. Some K562 cells without treatment were used as normal controls. 48 hours after the transfection Western blotting was used to detect the expression of P210bcr-abl fusion protein. 3H-TdR incorporation was used to detect the proliferation activity of K562. Annexin V-fluorescencein isothiocyanate (FITC)/phosphatidylinositol (PI) staining was used to detect the apoptosis of K2562 cells. Flow cytometry was used to observe the cell cycle of K562 cells. Benzidine staining was used to detect the differentiation of K562 cells towards erythrocytic series. Western blotting was used further to detect the expression of apoptosis-related protein Bcl-xL/Bax. Results (1) In contrast with the control groups, the expression level of bcr-abl chimeric gene was much lower in the RNAi group. (2) 3H-TdR incorporation test showed time-dependent inhibition of proliferation of K562 cells, reflected in decrease of counts per minute (CPM) value in RNAi group 24 h, 48 h, 72 h, and 96 h after siRNA transfection by 33.06%,52.25%,57.64%,and 70.87% respectively (F=17.7,P<0.01). (3) About 43.2% of K562 cells in the RNAi group were apoptotic 48h after siRNA transfection (F=13.6,P<0.01 ). (4) In contrast with the control groups, the expression of apoptosis-associated protein Bcl-xL was greatly down-regulated; however, the expression of Bax protein showed little change. (5) The percentage of benzidine-positive cells in the RNA

关 键 词：K562细胞 BCR-ABL融合基因 表达 特异性 Bcl-xL BLOT法 慢性粒细胞白血病 RNA干涉 小干涉RNA RNA分子 

分 类 号：R733.7[医药卫生—肿瘤]