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目的 探讨多单位核酶体外净化慢性粒细胞白血病 (慢粒 )骨髓的可能性 ,研究多单位核酶的体外切割活性及对慢粒细胞恶性表型的逆转作用。方法 针对慢粒发病中起重要作用的bcr abl融合基因 ,在融合位点两侧 4 4碱基范围内设计、合成 3个核酶 ,其中 2个切割位点位于bcr基因 ,1个位于abl基因。通过基因重组构建多单位核酶体外转录载体及逆转录病毒表达载体 ,鉴定其体外切割活性。将多单位核酶逆转录病毒表达载体转染K5 6 2细胞 ,应用MTT、3 H TdR掺入、RT PCR、斑点杂交、流式细胞仪、透射及扫描电镜等方法 ,检测多单位核酶对慢粒细胞增殖活性及细胞超微结构、细胞周期、凋亡等的影响。结果 多单位核酶的体外切割活性为 70 .8% ,逆转录病毒载体转染慢粒K5 6 2细胞系后 ,细胞DNA合成及细胞增殖受到明显抑制 ,转染后 96h抑制率达 5 0 %左右。多单位核酶转染细胞后能够有效切割细胞RNA ,使转染细胞RNA减少 10 0 0倍左右。流式细胞仪检测显示多单位核酶转染 72h后 ,18.4 %的细胞发生凋亡 ,大多数细胞被阻滞于G期 ,分裂期 (S期 )细胞数减少约 4 1.9%。透射、扫描电镜见转染细胞出现核固缩、凋亡小体等细胞凋亡的特异性表现。结论 多单位核酶不仅具有较高的体外切割活性 ,而且其逆转录病毒载体能够有效转染慢?
Objective To investigate the possibility of purification of chronic myeloid leukemia (CML) bone marrow by multi-unit ribozyme in vitro and to study the in vitro cleavage activity of multi-unit ribozyme and its reversal effect on malignant phenotype of chronic granulocyte. Methods Aiming at the bcr abl fusion gene that plays an important role in the pathogenesis of chronic granulositis, three ribozymes were designed and synthesized in 44 bases on both sides of the fusion site, of which two were located in the bcr gene and one in the abl gene . Multi-unit ribozyme in vitro transcription vector and retroviral expression vector were constructed by gene recombination to identify its in vitro cleavage activity. The multi-unit ribozyme retroviral vector was transfected into K562 cells, and MTT assay, 3 H TdR incorporation, RT PCR, dot blot, flow cytometry, transmission and scanning electron microscopy CGRP, cell ultrastructure, cell cycle, apoptosis and so on. Results The in vitro cleavage activity of multi-unit ribozyme was 70.8%. After retroviral vector was transfected into K562 cell line, the DNA synthesis and cell proliferation were significantly inhibited. After 96 h transfection, the inhibitory rate reached 50% about. Multi-unit ribozyme transfected cells can effectively cut cell RNA, transfected cells to reduce the RNA about 10 0 times. Flow cytometry showed that 72.4% of multi-ribozyme transfected cells were apoptotic, most of them were arrested in G phase, and the number of cells in S phase decreased by about 4 1.9%. Transfection, scanning electron microscopy showed that the transfected cells nuclear pyknosis, apoptotic bodies such as apoptosis-specific performance. Conclusion The multi-unit ribozyme not only has higher in vitro cleavage activity, but also its retroviral vector can be effectively transfected slowly?