Objective: To study Fas expression regulation of cytotoxic T lymphocyte(CTL)via anti-Fas ribozyme, increasing of CD80 epitope on the surface of acute myelomonocytic leukemia cells by CD80-IgG fusion protein and their effects on the apoptosis and killing ability against acute myelomonocytic leukemia cells of CTL.
Methods: A hammerhead ribozyme gene targeting the Fas mRNA was synthesized and its expression vector pEGFP-RZ596 was constructed and transfected into the mouse spleen T cells via electroporation. The Fas expression on T cells was detected by RT-PCR and Western bloting. In the meantime the eukaryotic expression vector pcDNA/CD80-IgG was constructed by gene recombinant technique and transfected into ovarian cells of hamster of the line CHO. The CD80-IgG fusion protein was purified from the supernatant of G418-selected CHO cells by Protein G affinity chromatography method. Then allogeneic mixed lymphocytes culture between the mouse spleen T cells transfected with pEGFP-RZ596 and WEHI-3 cells (mouse acute myelomonocyte leukemia cell line) incubated with CD80-IgG fusion protein was performed. The apoptosis rate of the T cells was detected with annexin V-FITC. The proliferation and killing ability in vitro against WEHI-3 cells of the T cells were detected by MTT colorimetry.
Results: The luminance of Fas Western bloting results from the mouse spleen T cells negative control, transfected with pEGFPC1 and transfected with pEGFP-RZ596 were separately 1, 0.98 and 0.45 (P < 0.01). After being cocultured with WEHI-3 cells, which has higher expression of Fas ligand (64% +/- 3%), the apoptosis rate and the killing ability against WEHI-3 cells of the mouse spleen T cells transfected with pEGFP-RZ596 were separately 37% and 67%. Whereas that of the mouse spleen T cells negative control and transfected with pEGFPC1 were separately 88%, 84% (P < 0.01) and 32%, 31% (P <0.01). The CD80 positive expression rate of WEHI-3 cells was upregulated from 5.1% +/- 0.4% to 27.4% +/- 2.2% after these cells were preincubated with CD80-IgG fusion protein (P < 0.01). The killing ability of the mouse spleen T cells against WEHI-3 cells preincubated and not preincubated with CD80-IgG fusion protein were separately 64% and 49% (P <0.01), but that of the mouse spleen T cells, which were transfected with pEGFP-RZ596, was further promoted to 82% (P < 0.01)
Conclusion: The apoptosis of mouse CTL inducing by FasL-Fas pathway could be avoided and the killing ability of mouse CTL against WEHI-3 cells can be significantly promoted at the same time by combining anti-Fas ribozyme and CD80-IgG fusion protein.