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目的进一步研究ｂｃｒ／ａｂｌ融合基因在慢性粒细胞白血病（ＣＭＬ）中作用以及探索ＣＭＬ基因治疗的可能性。方法利用ＤＮＡ重组技术将所设计、合成的针对ｂｃｒ／ａｂｌ融合转录本ｂ３ａ２断裂点“锤头型”核酶的基因用ＨＩｎｄⅢ、ｐｓｔⅠ酶切后克隆到ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ±质粒相应酶切位点获得ｐＢＲＺ重组子。测定ＲＺ基因序列与设计的一致。用ＢａｍＨＩ和ＸｈｏＩ从ｐＢＲＺ上切下ＲＺ基因，定向克隆到逆转录病毒载体ＰＬＸＳＮ，得到携带ＲＺ基因的重组逆转录病毒载体ＰＬＲＺＸＳＮ。通过脂质体介导的ＤＮＡ转染法，将ＰＬＲＺＸＳＮ导入慢性粒细胞白血病细胞株Ｋ５６２细胞，通过克隆分析法、ＲＴ－ＰＣＲ、流式细胞仪、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４８ｈ后，Ｋ５６２细胞克隆抑制率达８５％；（２）核酶转染７２ｈ后Ｋ５６２细胞Ｐ２１０ｂｃｒ—ａｂｌ蛋白表达率比未转染组低５２％；（３）核酶转染４８ｈ后，Ｋ５６２细胞ｂｃｒ／ａｂｌｍＲＮＡ表达水平比未转染组低７３％；（４）核酶处理的Ｋ５６２细胞发生凋亡，表现为核酶转染Ｋ５６２细胞７２ｈ后，用流式细胞仪可检测到明显凋亡峰，凋亡率为３７．１％，而空载体及脂?
Objective To further investigate the role of bcr/abl fusion gene in chronic myelogenous leukemia (CML) and explore the possibility of CML gene therapy. Methods The gene designed and synthesized for the bcr/abl fusion transcript b3a2 breakpoint “hammerhead” ribozyme was digested with HIndIII and pstI using DNA recombination technology and cloned into pBluescriptIISK± plasmid corresponding restriction site to obtain pBRZ recombination. child. The determination of the RZ gene sequence was consistent with the design. The RZ gene was excised from pBRZ using BamHI and XhoI, and cloned into the retroviral vector PLXSN, resulting in the recombinant retroviral vector PLRZXSN carrying the RZ gene. PLRZXSN was transfected into chronic myeloid leukemia cell line K562 cells by lipofectamine-mediated DNA transfection. The ribozyme was detected by clonality analysis, RT-PCR, flow cytometry, DNA electrophoresis and electron microscopy. The effect of the cells. Results (1) After 48h of ribozyme transfection, the inhibition rate of K562 cells reached 85%; (2) The expression rate of P210bcr-abl protein in K562 cells was 52% lower than that in non-transfection group after 72h of ribozyme transfection; (3) Nuclear After 48h of transfection, bcr/abl mRNA expression in K562 cells was 73% lower than that in non-transfected group; (4) Apoptosis was observed in ribozyme-treated K562 cells, which was expressed as ribozymes after transfecting K562 cells for 72h, using flow cytometry The instrument can detect obvious apoptotic peaks, with apoptotic rate of 37.1%, and empty carriers and lipids.