Transcription factor SP1 decoy ODNs targeting α1, 3-GT renders porcine endothelial cells resistant to complement-mediated cytotoxicity / 中华器官移植杂志

ABSTRACT

Objective To investigate whether porcine endothelial cells transfected with SP1 de-coy ODNs could resist complement-mediated cytotoxicity during the model of SV-40-PED with human serum in vitro. Methods Immortalized porcine aortic endothelial cells of the line PED were divided in-to three groups. The transfected group was SV-40-PED with SP1 decoy ODNs. The mismatched group was SV-40-PED with scrambled SP1 decoy ODNs. The negative group was cells with oligo-fectamine only. The expression of α1,3-GT mRNA and αGal was detected after 26 h by using fluores-cence microscope, Western blot, RT-PCR and lactate dehydrogenase (LDH) activity assay. Results Fluorescence microscopy observed the decreased fluorescence of αGal after SP1 decoy ODNs transfec-tion. Dotted fluorescent decrease could be observed on some membrane while the mismatched group and negative group with bright green fluorescence. Western blot showed that the average absorbance of the PED cells transfected with decoy ODNs was decreased to 52.6% of the negative group (P<0.05). The expression of α1,3-GT mRNA in the PED cells transfected with decoy ODNs was 0.42±0.20 (isoform 1) and 0.27±0.12 (isoform 2) respectively, significantly lower than in the negative group (isoform 1, 0.72±0.17; isoform 2, 0.50±0.19; both P<0.05). The expression of α1, 3-G Tin the mismatch group was not different from that in the negative group (P>0.05). 20% normal hu-man serum (NHS) and 40 % NHS had cytotoxic effect on both mismatch and negative groups, but de-coy ODNs could confer SV-40-PED protection from the cytolysis effect (P<0.05), which made a re-markable reduction of complement-mediated cytotoxicity towards SV-40--PED. Conclusions SP1 decoy ODNs could confer porcine endothelial cells protection from complement-mediated cytotoxicity effect in vitro.