Abstract: Objective To evaluate the effect of RNA interference (RNAi) targeting pig alpha 1,3-galactosyltransferase (α1,3-GT) on the human natural killer cell-induced xeno-cytotoxicity.Methods The immortalized pig endothelial cell line (PED) was subgrouped into transfected (SiRNA-1),mock and scrambled control groups.The effect of SiRNA-1 on the interaction between PED and human natural killer cell line (NK92) was assessed in terms of adhesion and cytotoxicity,as well as IFN-γ levels of the coculture supernatant of PED and NK 92.Results Forty-eight h post-transfection of SiRNA-1 into PED,there was no significant difference in the adhesion of NK 92 to PED under static or dynamic condition among SiRNA-1,scrambled and mock groups.The number of NK 92 adhering to PED was 262±26,275±24 and 252±23 under static condition and 132±12,125±15 and 110±20 under dynamic condition in SiRNA-1.scrambled and mock groups,respectively (P>0.05).Direct cytotoxicity of NK 92 towards PED in all groups was almost the same with or without PED activation and exhibited a positive correlation with the ratio of effector to target cells.There was induced IFN-γ secretion in supernatant at the early period of co-culture [(366.9±28.7)ng/L],resulting in a five-fold rise compared to spontaneous secretion [(60.1±6.4)ng/L].The maximum secretion level was at 12 h post-transfection and reduced subsequently.There was no statistically significant difference at various time points among SiRNA-1 transfected,scrambled transfected and mock groups (P>0.05).Conclusions There was no significant change in the interaction of NK 92 and PED after knock-down expression of α-Gal by RNAi,indicating that α-Gal might not the target site of hNK cytotoxicity.The level of α-Gal expression was not related with hNK cells activation,adhesion and cytotoxicity.