摘要
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.
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.
基金
National Natural Science Foundation of China (No. 39670330).