摘要
目的:探讨伊马替尼和P27基因克隆联合作用对K562细胞的细胞增殖、细胞周期及凋亡等的调控作用。方法:RT-PCR扩增P27基因,胶纯化回收后连接到T载体测序,序列正确后构建P27-pcDNA3.1载体,将P27-pcDNA3.1与空载体分别以脂质体转染入P27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P27蛋白表达,MTT法检测细胞生长指数和细胞存活率,流式细胞仪检测细胞周期和凋亡。结果:表达外源性P27蛋白的P27-pcDNA3.1-K562细胞株生长速度明显慢于对照K562细胞株,流式细胞仪显示G0/G1期细胞增多,S期细胞明显减少,P27-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,MTT法显示细胞存活率较P27-K562细胞组及伊马替尼-K562细胞组均明显下降(P<0.01vsP27-K562细胞组,P<0.05vs伊马替尼-K562细胞组)。结论:表达外源P27蛋白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 effect of imatinib combined with P27 gene clone on the proliferation, cycle and apoptosis of chronic myeloid leukemia cell line K562. Methods P27 gene was amplified from peripheral blood mononuclear cells by RT-PCR, and its sequences were confirmed. P27-pcDNA3.1 vector was constructed and then transfected into P27 gene-deleted K562 cell line by lipofectine. After screened with G418, P27-pcDNA3. 1-K562 cell clone that stably expressed P27 was isolated. P27 protein was identified by Western blot. The cell survival rate was tested by MTT, cell cycle and apoptosis were tested by flow cytometry. Results The expression of P27 protein could be detected by Western blot in P27-pcDN3. 1-K562 cells. A strong inhibition of cell proliferation was observed in P27-pcDNA3. 1-K562 cell compared with the control K562 cell. P27-pcDNA3. 1-K562 cells increased apparently in G0/G1 phase but declined greatly in S phase. Cell cycle was arrested in G0/ G1 phase. The pereentage of apoptosis greatly increased after P27-pcDNA3. 1-K562 cells were combined with imatinib and the cell survival rate greatly declined. Conclusion Imatinib combined with P27 gene clone has a synergetic effect on inhibiting cell proliferation and promoting apoptosis of K562 cell.
出处
《实用医学杂志》
CAS
2006年第13期1473-1476,共4页
The Journal of Practical Medicine
基金
国家自然科学基金项目(编号:39770336)
关键词
基因产物
rex
哌嗪类
克隆
分子
增殖抑制
凋亡
Gene product, rex Piperazines Cloning, molecular Proliferation inhibition Apoptosis
