[Construction of retrovirus vector of bcr/abl mRNA cleaving ribozyme gene and its effects on K562 cells].

OBJECTIVE

To investigate the effect of bcr/abl fusion gene on the growth of chronic myeloid leukemia(CML) cells and to explore the feasibility of ribozyme in CML gene therapy.

METHODS

A hammerhead ribozyme DNA targeting the bcr/abl (b3a2) transcript was synthesized, and recombinated into retroviral vector pLXSN forming pLRZXSN recon. By lipofectin mediated DNA transfection technique, pLRZXSN was introduced into K562 cells. The effects of the ribozyme on the growth of K562 cells and apoptosis were studied by leukemic colony assay, flow cytometry (FCM), reverse transcript-polymerase chain reaction (RT-PCR), detection of DNA fragmentation and electron microscope.

RESULTS

1. The number of K562 cell colony was inhibited by 85% after the cells were transfected for 48 hours. 2. The expression of bcr/abl mRNA and the fusion protein P210 was decreased sharply in K562 cells transfected with pLRZXSN for 48 and 72 hours, respectively. 3. The characteristics of apoptosis was revealed in K562 cells transfected with pLRZXSN, i.e. sub-G1 peak, DNA fragmentation and morphological changes.

CONCLUSION

The ribozyme was capable of inhibiting the proliferation of K562 cells and inducing the cell apoptosis.