摘要
Objective:To generate an h MSH2-gene knockdown lung cancer cell model with small RNA interference in NCI-H520 cell line,so as to verify the underlying mechanisms in h MSH2-mediated recognition and cytolyisis of lung cancer cells by Vγ9δ2 T cells.Methods:h MSH2 specific si RNAs were designed,synthesized and transfected into NCI-H520 cells.Gene knockdown efficiency was measured by q RT-PCR,Westernblot,flow cytometery and confocal microscopy 48 or 72 h after si RNAs transfection.Results:Transfection efficiency was up to 93% 6 h after si RNAs treatment.48 or 72 h after specific si RNAs transfection,si RNA duplex I and II respectively resulted in 97%,88% and 98%,97% reduction in h MSH2 m RNA expression when compared to mock control.Reduced h MSH2 protein expression was confirmed by Western blot.Decreased surface expression of h MSH2 in si RNA-treated groups was revealed by flow cytometery and confocal microscopy.Conclusion:si RNA-h MSH2 gene knockdown lung cancer cell model was successfully generated in NCI-H520 cell line,providing a useful means to explore the recognition and cytolytic mechanism in Vγ9δ2 T cell-mediated anti-lung cancer innate immunity.
Objective:To generate an h MSH2-gene knockdown lung cancer cell model with small RNA interference in NCI-H520 cell line,so as to verify the underlying mechanisms in h MSH2-mediated recognition and cytolyisis of lung cancer cells by Vγ9δ2 T cells.Methods:h MSH2 specific si RNAs were designed,synthesized and transfected into NCI-H520 cells.Gene knockdown efficiency was measured by q RT-PCR,Westernblot,flow cytometery and confocal microscopy 48 or 72 h after si RNAs transfection.Results:Transfection efficiency was up to 93% 6 h after si RNAs treatment.48 or 72 h after specific si RNAs transfection,si RNA duplex I and II respectively resulted in 97%,88% and 98%,97% reduction in h MSH2 m RNA expression when compared to mock control.Reduced h MSH2 protein expression was confirmed by Western blot.Decreased surface expression of h MSH2 in si RNA-treated groups was revealed by flow cytometery and confocal microscopy.Conclusion:si RNA-h MSH2 gene knockdown lung cancer cell model was successfully generated in NCI-H520 cell line,providing a useful means to explore the recognition and cytolytic mechanism in Vγ9δ2 T cell-mediated anti-lung cancer innate immunity.
引文