Antisense SP1 Oligodeoxynucleotides And SP1 Decoy Oligodeoxynucleotides Mediated By Oligofectamine Suppress Xenogeneic Response In Porcine Aortic Endothelial Cells Targeting Promoter B Of α1,3 Galactosyltransferase

PartⅠOptimizing condition for oligofectamine-mediated SP1 antisense and decoy oligodeoxynucleotides transfection into SV-40-PED cells【Objective】To determine the optimizing parameters in transfecting SV-40-PED cells mediated by oligofectamine so as to provide useful information for the targeting transcription factor on suppressing xenograft rejection.【Methods】AS-ODN ,decoy ODN and mismatch ODN were bio-synthesized in vitro targeting promoter B of upstream regulation region ofα1,3 galactosyltransferase (α1,3-GT) gene of porcine. After delivering these ODNs into SV-40-PED by oligofectamine transfection system, the uptake rate and mean fluorescence intensity of ODNs were measured by flow cytometry to evaluate transfection efficiencies. Intracellular distribution of ODNs was determined with fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was assayed to assess the cytotoxicity.【Results】The uptake of ODNs into SV-40-PED cells was significantly improved by oligofectamine. In 6 well culture plate, 4μl ODNs with 12.5μl oligofectamine resulted in the highest transfection efficiency and lower cytotoxicity( LDH in the supernatant was 49.61±17.13 U/L). The uptake of ODNs was 93.37±0.85%,the mean fluorescence was 200.30±12.22. The ODNs localized in the nucleus following an incubation of 26 h with oligofectamine. There is no difference between the mismatch group and negative control group.【Conclusion】Oligofectamine transfection system was a high efficient and low toxic tool to deliver oligodeoxynucleotides into target cells. In 6 well culture plate, 4μl ODNs with 12.5μl oligofectamine resulted in the highest transfection efficiency and lower cytotoxicity. The ODNs localized in the nucleus following an incubation of 26 h with oligofectamine. This study made a foundation for the targeting transcription factor on suppressing xenograft rejection. PartⅡThe effect of transcription factor SP1 antisense and decoy oligodeoxynucleotides on expression ofαGal in SV-40-PED cells【Objective】To investigate the role of transcription factor SP1 antisense and decoy oligodeoxynucleotides on expression ofα1,3-GT gene andαGal proteins in SV-40-PED cells.【Methods】Immortalized porcine aortic endothelial cells of the line PED were cultured and transfected withα1, 3galactosyltransferase (α1,3GT) specific antisense and decoy ODNs. Cells transfected with mismatch ODNs was used as negative controls. Twenty-six hours later the cells were collected. The expression ofαGal was determined with fluorescence microscope, Western blot and flow cytometry. The expression ofα1,3GT mRNA was examined by RT-PCR.【Results】Fluorescence microscopy observed the decreased fluorescence ofαGal after decoy ODNs transfection. Western blot showed that the average absorbance of the PED cells transfected with decoy ODNs was (48.2±0.9).It is 52.6% of the mock group(P < 0 .05). The expression ofα1,3GT mRNA in the PED cells transfected with decoy ODNs was (isoform 1,0.42±0.20;isoform 2,0.27±0.12) lower in comparison with the mock group(isoform 1,0.72±0.17;isoform 2,0.50±0.19;both P < 0.05).The expression ofα1, 3GT in the mismatch group was not different from those in the mock group (P >0.05) .【Conclusion】α1,3GT gene reduce actually occurs following transfection of decoy ODNs. Porcine endothelial cells can be the targets of decoy ODNs, which providing new ideas and strategy for the research of inhibiting hyperacute rejection. PartⅢSP1 decoy ODNs targetingα1,3GT renders porcine endothelial cells resistant to complement-mediated cytotoxicity【Objective】To investigate whether porcine endothelial cells transfering with decoy ODNs could resist complement mediated cytotoxicity during a model of SV-40-PED with human serum in vitro.【Methods】The influence of down-regulatedα-Gal expression on the antibodies and complement binding to SV-40-PED was observed by flow cytometry. The effect of decoy ODNs transfering into porcine endothelial cells protection from complement-mediated cytotoxicity was assessed by lactate dehydrogenase (LDH) activity assay.【Results】The binding of antibodies and complement to SV-40-PED in decoy ODNs group decreased to various extent compared to mock group. The percent reduction of antibodies and complement binding to SV-40-PED was 68.0% (IgM), 56.1% (IgG) and 55.1% (C3c) (P < 0.05), respectively. 20% normal human serum (NHS) and 40% NHS had cytotoxic effect on both scrambled and mock groups, but decoy ODNs could confer SV-40-PED protection from the cytolysis effect. The rate of complement-mediated cytotoxicity decreased 15.0% in 20% NHS and 30.2% in 40% NHS (P <0.05) compared with the mock group, which made a remarkable reduction of complement-mediated cytotoxicity towards SV-40-PED. The lysis rate between mock and scrambled groups had not significant difference (P >0.05). HINHS displayed only background cytotoxic activity to all SV-40-PED cells due to no complement activity.【Conclusion】Decoy ODNs could confer porcine endothelial cells protection from complement-mediated cytotoxicity effect during the model of SV-40-PED with human serum in vitro, which providing new ideas and strategy for the research of overcoming hyperacute rejection of pig-to-human xenografts. PartⅣEffect of SP1 decoy ODNs targetingα1,3GT on the interaction between porcine endothelium and human peripheral blood mononuclear cell【Objective】To evaluate the effect of SP1 decoy ODNs transfering into SV-40-PED during a co-culture model between porcine endothelial cells and human peripheral blood mononuclear cells in vitro and observe the possible protection to PEC by decoy ODNs.【Methods】The influence of down-regulatedα-Gal expression on the interaction between porcine endothelial cells and human peripheral blood mononuclear cells (PBMC) was evaluated by 3H-thymidine (3H-TdR) incorporation and MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay.【Results】There was a weak proliferation in the purely human peripheral blood mononuclear cells group. After co-culturing with stimulator SV-40-PED which were treated with mitomycin, cell proliferation was observed in each group. The number of surviving cells achieved maximum slowly at the 5-7 day. At the day of fifth, the cpm of the decoy ODNs group was decreased. It is 67.1% of the mock group(P < 0 .05). The uptake of 3H-TdR between mock and scrambled groups had not significant difference (P >0.05). The inhibition rate of reproductive activity of human peripheral blood mononuclear cells was 39.2% by MTT assay in decoy ODNs group (P <0.05). The proliferation rate between mock and scrambled groups had not significant difference (P >0.05).【Conclusion】In the co-culture model between porcine endothelial cells and human peripheral blood mononuclear cells in vitro, proliferation of human peripheral blood mononuclear cells can be inhibited partly by transfering decoy ODNs into SV-40-PED.