Study On The Proapoptotic Effect Of A Ribozyme Against Bcl-2 On HL 60 Cells

In this experiment, a ribozyme(RZ) targeted to bcl-2 mRNA was constructed and the retroviral vector, pDOR-neo was used to express this RZ in HL 60 cells. After analysis of the secondary structure of bcl-2 mRNA by computer, a hammerhead ribozyme for cleaving bcl-2 mRNA at a specific site between 1656 C and 1657 G was designed and its gene (RZ DNA) synthesized with an automatic DNA synthesizer. The two complementary strands of RZ DNA was annealed and cloned into the Hinc II site of pGEM-3Zf(-), and the sequence of RZ DNA insert in this recombinant plasmid [named as 3ZRZ(+)/(-)] was determined and proved to be perfectly correct. Both RZ DNA and bcl-2 cDNA were transcribed in vitro, forming the ribozyme and bcl-2 mRNA, respectively, with the latter radio-labeled. When mixing the two transcripts under 50 ℃ for 2h, the ribozyme showed a strong catalytic activity in site-specific cleavage ofbcl-2 mRNA. The RZ DNA was then cut down from 3ZRZ(-) and subcloned orientally into the corresponding site of the eukaryotic expression vector pDOR-neo to form a recombinant retroviral vector pDOR-RZ.pDOR-RZ was successfully introduced into HL 60 cells by lipofectin-mediated DNA transfection and the cells were maintained in culture for 72h. The expression of RZ was observed by RNA dot blot and in situ hybridization. The effects of RZ on the growth and apoptosis of HL 60 cells were studied by flow cytometry (FCM), electron microscopy, immunocytochemistry of Bcl-2 protein and agarose gel electrophoresis...