β-TrCP-OD-HA重组腺病毒对白血病K562细胞凋亡的影响  

Effect of β-TrCP-OD-HA recombinant adenoviruses on apoptosis of leukemia K562 cells

作　　者：罗红伟[1] 田文君[1] 季茂胜[1] 刘钉宾[1] 王小飞[1] 黄宗干[2] 冯文莉[1] 

机构地区：[1]重庆医科大学:临床血液学教研室,临床检验诊断学教育部重点实验室,重庆400016 [2]重庆医科大学附属第一医院血液科,重庆400016

出　　处：《第三军医大学学报》2010年第15期1602-1604,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金(30670901)~~

摘　　要：目的研究癌蛋白Bcr-Abl被泛素-蛋白酶体系统靶向降解的可能性及诱导白血病K562细胞凋亡的作用。方法含有Bcr-Abl融合蛋白N端寡聚化区(OD)的重组腺病毒Ad5β-TrCP-OD-HA特异性结合Bcr-Abl融合蛋白的N端寡聚化区(OD),通过感染将嵌合泛素连接酶β-TrCP运送到靶细胞K562细胞,同时,β-TrCP基因突变的重组腺病毒Ad5△F-TrCP-OD-HA和仅含绿色荧光蛋白基因的重组腺病毒Ad5GFP,分别作为对照。感染后,Western blot检测Bcr-Abl融合蛋白的表达;倒置显微镜、光镜和电镜观察细胞形态学变化。结果成功建立携β-TrCP-OD-HA、△F-TrCP-OD-HA的重组腺病毒的K562细胞株,重组腺病毒感染K562细胞后,Ad5β-TrCP-OD-HA组的Bcr-Abl融合蛋白含量下降,与Ad5β-TrCP-OD-HA组、Ad5GFP组及对照组相比,差异具有统计学意义(P<0.05);倒置显微镜可见Ad5β-TrCP-OD-HA作用后细胞体积变小、形态不规则;光镜下发现胞体变小,细胞质空泡,细胞核出现碎裂和凋亡小体;电镜观察到出现了典型的细胞凋亡的超微结构。结论重组腺病毒介导的β-TrCP-OD-HA在白血病K562细胞内能够降低Bcr-Abl融合蛋白含量,并且诱导K562细胞发生了凋亡。Objective To study whether the oncoprotein, Bcr-Abl, can be degraded by ubiquitin- proteasome target system and its role in induction of apoptosis of leukemia K562 cells. Methods Chimeric ubiquitin ligase, β-TrCP, was transported to target K562 cells by Ad5β-TrCP-OD-HA recombinant adenoviru- ses carrying N-terminal oligomerizaton domain （OD） of Bcr-Abl that can specifically bind to its N-terminal OD through infection. Meanwhile, recombinant adenovirnses with mutational β-TrCP gene （Ad5ΔF-TrCP-OD-HA） and green fluorescent protein gene（Ad5GFP）were used as controls, respectively. After infection, expression of Ber-Abl was detected by Western blotting. Cell morphology was observed under inverted, light and electron microscopes, respectively. Results The leukemia K562 cell clones with exogenous recombinant β-TrCP-OD-HA or ΔF-TrCP-OD-HA gene were established. After infection with recombinant viruses, the expression level of Bcr-Abl was lower in Ad5β-TrCP-OD-HA than in Ad5GFP and contruls （P 〈 0. 05）. Inverted microscopy demonstrated small-sized ceils in irregular shape, light microscopy showed small-sized cells, vacuolation in cytoplasm, cleavage of nuclei, and apoptotic bodies, and electron microscopy revealed typical ultrastructure of apoptotic cells, after treatment with Ad513-TrCP-OD-HA. Conclusion Recombinant adenoviruses-indueed β-TrCP-OD-HA can reduce Bcr-Ahl in leukemia K562 cells and induce their apoptosis.

关 键 词：Bcr—Abl融合蛋白 泛素连接酶 β-TrCP K562细胞 细胞凋亡 

分 类 号：R394-33[医药卫生—医学遗传学] R73-362[医药卫生—基础医学]