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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 role of bcr / abl fusion gene in chronic myeloid leukemia (CML) and explore the possibility of gene therapy for CML. METHODS: The cDNA sequence of “hammerhead” ribozyme targeting bcr / abl fusion gene transcript b3a2 was synthesized and cloned into a retroviral vector. The ribozyme gene K562 cells were transfected with K562 cells. The effects of ribozyme on K562 cells were detected by cloning analysis, flow cytometry (FCM), reverse transcription-polymerase chain reaction (RT-PCR), DNA electrophoresis and electron microscopy. Results: ①The inhibition rate of cloning formation reached 48% at 48 hours after transfection with K562 cells; ② The synthesis of P210 protein in cells was significantly inhibited; ③ The expression of bcr / abl mRNA was significantly decreased by RT-PCR; ④ The expression of K562 Apoptosis occurred in the cells, showing obvious apoptotic peak on the FCM map; DNA ladder analysis showed a typical DNA ladder; electron microscopy showed early apoptotic morphological changes. CONCLUSION: The ribozyme gene can be successfully expressed in K562 cells by retroviral vector. The expression of bcr / abl mRNA and P210 protein in K562 cells is decreased, which inhibits the proliferation of K562 cells and induces the apoptosis of K562 cells.