p16基因克隆联合伊马替尼对慢性粒细胞白血病K562细胞作用的研究  

Effect of p16 gene cloning combined with imatinib on chronic myeloid leukemia K562 cell line

作　　者：王玮[1] 孙秉中[2] 刘庆春[1] 药立波[3] 

机构地区：[1]解放军第175医院血液肿瘤科,福建省漳州市363000 [2]第四军医大学西京医院血液科,西安市710032 [3]第四军医大学生物化学和分子生物学教研室,西安市710032

出　　处：《实用医学杂志》2008年第5期700-703,共4页The Journal of Practical Medicine

基　　金：国家自然科学基金资助项目(编号:39770336)

摘　　要：目的:探讨p16基因克隆联合伊马替尼对慢性粒细胞白血病细胞株K562细胞的增殖、周期及凋亡等的调控作用。方法:RT-PCR扩增p16基因,胶纯化回收后连接到T载体测序,序列正确后构建p16-pcDNA3.1载体,将p16-pcDNA3.1与空载体分别以脂质体转染入p16基因缺失的K562细胞,经筛选得到G418抗性的K562细胞株,Westernblot证实转染后有p16蛋白表达,MTT法检测细胞存活率,流式细胞仪检测细胞周期和凋亡。结果:反复测序发现,从K562细胞中扩增的DNA片段属于非特异性扩增,证实K562细胞中p16基因缺失。转染p16基因后表达外源性p16蛋白的p16-pcDNA3.1-K562细胞株生长速度明显减慢;伊马替尼作用于已转染p16-pcDNA3.1的K562细胞,其细胞存活率与伊马替尼作用未转染K562细胞及单纯转染p16-pcDNA3.1的K562细胞相比均明显降低。转染p16基因后G0/G1期细胞增多,S期细胞明显减少,p16-pcDNA3.1-K562细胞与伊马替尼联合应用后凋亡细胞比例明显上升。结论:p16基因抑制K562细胞增殖并调节其周期,外源p16联合应用伊马替尼对抑制K562细胞增殖及促凋亡方面具有协同作用。Objective To explore the regulatory effect of p16 gene cloning combined with imatinib on the proliferation, cycle and apoptosis of chronic myeloid leukemia K562 cell line, Methods p16 gene was amplified by RT- PCR and purified by gel and then bound to vector T to be sequenced, Vector pl6-pcDNA3,1 was constructed after the exact sequences were obtained, and transfected into the K562 cells with p16 gene deletion by liposome, After being selected with G418, the G418-resistant p16-pcDNA3,1-K562 cells were isolated, The expressions of p16 protein were detectable by Western blot. The survival rate of the cell was tested by MTY and the cell cycle and apoptosis by flow cytometry, Results Repeated sequencing revealed the DNA fragments amplified from K562 cell line was nonspecific amplification, confirming p16 gene deletion in K562 cell line. The growth of p16-pcDNA3.1-K562 cell line expressing p16 protein was evidently declined after transfection of p16 gene, The survival rate of p16-pcDNA3.1-K562 cells affected by imatinib was significantly lower than that with no imatinib affection and that of K562 cells. The pl6-pcDNA3,1-K562 cells increased in G0/G1 phase and declined markedly in S phase in which the apoptotic rate increased obviously when combined with imatinib. Conchmions p16 gene inhibits the proliferation of K562 cells and regulates the cell cycle, Combination of p16 gene with imatinib has a synergistic effect on inhibiting the proliferation of the cells and enhancing their apoptosis.

关 键 词：白血病 淋巴细胞 慢性基因 P16 K562细胞伊马替尼 基因克隆 

分 类 号：R733.7[医药卫生—肿瘤]