摘要
目的：研究ｂｃｒ／ａｂｌ融合基因在慢性粒细胞白血病（ＣＭＬ）中的作用以及探索ＣＭＬ基因治疗的可能性。方法：合成针对ｂｃｒ／ａｂｌ融合基因转录本ｂ３ａ２断裂点“锤头状”核酶的ｃＤＮＡ序列，定向克隆于逆转录病毒载体内，通过脂质体介导的ＤＮＡ转染法，将核酶基因导入Ｋ５６２细胞，并通过克隆分析法、流式细胞术（ＦＣＭ）、逆转录聚合酶链反应（ＲＴＰＣＲ）、ＤＮＡ电泳及电镜观察等方法检测核酶对Ｋ５６２细胞的影响。结果：①核酶转染Ｋ５６２细胞后４８小时克隆形成抑制率达８５％；②细胞内Ｐ２１０蛋白合成受到明显抑制；③ＲＴＰＣＲ半定量检测ｂｃｒ／ａｂｌｍＲＮＡ表达水平明显下降；④核酶处理组Ｋ５６２细胞发生凋亡，表现为：在ＦＣＭ图谱上可见明显的凋亡峰；ＤＮＡ电泳分析出现典型的梯状ＤＮＡ带；电镜观察呈现凋亡早期形态学改变。结论：核酶基因通过逆转录病毒载体导入Ｋ５６２细胞后可成功表达，使Ｋ５６２细胞ｂｃｒ／ａｂｌｍＲＮＡ及Ｐ２１０蛋白表达水平下降，抑制Ｋ５６２细胞增殖，同时诱导Ｋ５６２细胞发生凋亡。
Objective: To investigate the effect of bcr/abl fusion gene on the growth of chronic myeloid leukemia(CML) cells and to explore the feasibility of ribozyme in CML gene therapy. Methods: A hammerhead ribozyme DNA targeting the bcr/abl (b3a2) transcript was synthesized, and recombinated into retroviral vector pLXSN forming pLRZXSN recon. By lipofectin mediated DNA transfection technique, pLRZXSN was introduced into K562 cells. The effects of the ribozyme on the growth of K562 cells and apoptosis were studied by leukemic colony assay,flow cytometry (FCM), reverse transcript polymerase chain reaction (RT PCR), detection of DNA fragmentation and electron microscope. Results: ①The number of K562 cell colony was inhibited by 85% after the cells were transfected for 48 hours.②The expression of bcr/abl mRNA and the fusion protein P210 was decreased sharply in K562 cells transfected with pLRZXSN for 48 and 72 hours, respectively. ③The characteristics of apoptosis was revealed in K562 cells transfected with pLRZXSN, i.e. sub G1 peak, DNA fragmentation and morphological changes. Conclusion: The ribozyme was capable of inhibiting the proliferation of K562 cells and inducing the cell apoptosis.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
1998年第12期623-626,共4页
Chinese Journal of Hematology
