Objective: To investigate the in vitro cleavage ability and effects on apoptosis and cell growth of the bcr-abl fusion gene specific multi-unit ribozymes. Methods: Three fusion point specific ribozymes were designed and the multi-unit ribozymes?in vitro transcription vector and retroviral vector were constructed. The in vitro cleavage ability was tested. The retroviral vector was transfected into K562 cell and the effects on proliferation, apoptosis, cell cycle and cell structure were observed. Results: Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%, which was more efficient than single-unit and double-unit ribozymes. Transfection of the retroviral vector of the ribozyme into K562 cells, induced inhibition of cell growth and apoptosis. The incorporation rate of DNA in ribozymes transfected K562 cells was greatly decreased along with time passed, with an inhibition rate of more than 50% after 96 h of transfection. Under FCM, 18.4% of the cells underwent apoptosis 72 h after transfection and more cells were blocked in G phase, with the ratio in S phase greatly decreased (41.9%). Under electron microscope, compaction of nuclear chromatin and apoptosis bodies were observed. Conclusion: Multi-unit ribozymes specific to bcr-abl fusion gene can be used to treat CML and to purge bone marrow for self-grafting.
目的分离K562细胞释放的exosomes，致敏脐血树突细胞（dendritic cell，DCs），观察其对细胞毒性T淋巴细胞（cytotoxic T lymphocytes，CTLs）的激活效应。方法离心超滤和蔗糖密度梯度离心法分离K562细胞释放的exosomes，固相免疫电镜法（SPIEM）制备exosomes的HSP70、ICAM-1及ABL免疫电镜标本。常规方法从脐血单个核细胞诱导DCs并分离T细胞，将K562细胞来源的exosomes冲击或未冲击的DCs与T细胞共培养。MTT比色法检测体外细胞毒活性。结果K562细胞分泌的exosomes为直径50～100nm的膜性微囊。Exosomes致敏的脐血DCs激活CTLs的能力显著高于肿瘤冻融抗原致敏的DCs组，在效靶比为50：1时，两组CTLs对l（562细胞的杀伤率为（68，4％vs35．3％，P〈0．05）。结论K562细胞分泌的exosomes负载脐血DCs后活化CTLs，有抗肿瘤活性。
