Title: Human ovarian carcinoma cells transfected with GM-CSF and IL-21 genes and its effect on the cytotoxic activity of NK cells
Abstract: Objective To explore the in vitro anti-tumor effect of cotransfection of GM-CSF and IL-21 genes. Methods The IL-21 gene was amplified from recombinant pRSC-IL21 by PCR and cloned directly into the recombinant pRSC-GM-CSF to establish a new recombinant plasmid, pRSC-GM-CSF-IL21. And then, pRSC-GM-CSF, pRSC-IL21, and pRSC-GM-CSF-IL21 were respectively transfected into SKOV3 cells by using lipofectamine (SKOV3/GM-CSF, SKOV3/IL21, and SKOV3/GM-CSF-IL21 groups). The proliferation activity of the transfected SKOV3 cells was determined using MTT method. The expressions of GM-CSF and IL-21 genes, cell surface molecular of human leucocyte antigen-ABC (HLA-ABC), MHC class-I-chain-related protein (MIC A/B), and intercellular adhesion molecule-1 (ICAM-1), and distribution of cell cycle were detected by RT-PCR and flow cytometry respectively. The effect of GM-CSF and IL-21 secreted by SKOV3 cells on the cytotoxicity of NK cells was also assayed. Inverted phase contrast microscope was employed to observe the morphology of the cells. Non-transfected SKOV3 cells (SKOV3 group) and the cells transfected with blank plasmid pRSC (SKOV3/Neo group) were set as controls. Results The recombinant pRSC-GM-CSF-IL21 plasmid was identified by endonuclease digestion and DNA sequencing. The expression of GM-CSF and IL-21 genes was detected in the SKOV3 cells after cotransfection. No significant differences among morphology, proliferation activity, distribution of cell cycle, and HLA-ABC expression were detected among the SKOV3, SKOV3/Neo, SKOV3/GM-CSF, SKOV3/IL21, and SKOV3/GM-CSF-IL21 cells. The expressios of MICA/B in the 5 groups of cells were 19.24% ± 2.31%, 31.11% ± 3.76%, 37.27% ± 3.04%, 54.97% ± 4.77%, and 63.94% ± 5.90%, respectively; the expression of ICAM-1 were 35.88% ± 3.06%, 37.75% ± 4.09%, 68.59% ± 4.84%, 78.53% ± 5.78%, and 84.10 ± 3.98%; and the NK cytotoxicity were 31.8% ± 3.6%, 42.7% ± 3.3%, 54.7% ± 7.4%, 55.9% ± 4.4%, and 63.4% ± 6.4% after been treated with the supernatant of the 5 groups (P 0.001 for all). Conclusion The expressions of MIC A/B and ICAM-1 on the surface of SKOV3 cells can be upregulated by cotransfection of GM-CSF and IL-21 gene, which can increase the cytotoxicity of NK cells, activating immune cells and enhancing anti-tumor effect.
Publication Year: 2008
Publication Date: 2008-01-01
Language: en
Type: article
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