| Lentiviral vectors with small haprpin RNA (shRNA) focusing on mouse a 1,3 galactosyltransferase (al,3GT) and RelA, a subunit gene of nuclear factor-kappa B (NF-κB), were constructed. The effects of RNA interference on al,3GT and RelA gene expression were observed by in vitro transfection of epithelium of mouse angioma (EOMA) cells and in vivo transfection of mouse heart. ShRNA lentiviruses with high performance against each gene were screen out by in vitro transfection of EOMA. Effects of in vivo transfection of mouse heart were performed by injection of lentivirus via vena dorsalis penis. Mouse-to-rat heterotopic cardiac xeno-transplantation model was constructed by using mouse heart with low al,3GT and RelA expression as the donor heart. The regulatory effects and mechanisms of lentivirus mediated RNA interference on al,3GT and RelA were discussed.Part I Construction of al,3GT and RelA lentiviral vectors and relative experiments in vitroObjective shRNA lentiviral vectors focus on mouse a 1,3 galactosyltransferase (al,3GT) and small hairpin RNA (shRNA) of RelA were constructed. In vitro transfection rate and inhibiting effects of shRNA lentiviral vectors were observed, and shRNA lentivrus vectors with high performance were screened.Methods 3 specifies shRNA focus on al,3GT and RelA mRNA were designed respectively, and high titer recombinated lentiviral vectors carrying al,3GT and RelA shRNA plasmids were constructed separately. In vitro transfections of EOMA were performed and conditions were optimized. Grouping as followed:Group 1 Blank control, Group 2 negative control, Group3-5 al,3GT-shRNA interfere group, Group 6-8 RelA-shRNA interfere group. QPCR and immunofluorescence were used to observe theα1,3GT, RelA, and Gal a-1,3-Gal antigen expression after transfection.Results Plasmids carrying al,3 GT and RelA shRNA were constructed, and high titer recombinated lentiviral vectors carrying al,3GT and RelA shRNA plasmids had a high and stable transfection rate on EOMA in vitro. Comparing with the blank and negative control, both al,3GT-shRNA and RelA-shRNA interfere groups showed a decrease in al,3GT and RelA expression (p<0.01). Galα1,3Gal antigen level of al, 3GT-shRNA interfere group showed a significant decrease when comparing with the blank and negative control group (p<0.01), within themα1,3GTi-3 and RelAi-3 shRNA interfere groups had the highest inhibition effects on the expression of the target gene.Conclusion Constructed al,3GT-shRNA and RelA-shRNA lentiviral vectors can highly and stably perform transfecton on EMOA in vitro.α1,3GT and RelA gene expressions and Galal,3Gal antigen expression are significantly depressed indicating thatα1,3GTi-3 and RelAi-3 shRNA lentiviral vectors are relatively high performed, which can be used for in vitro and animal experiments.PartⅡExperiment by injecting lentivirus vectors via vena dorsalis penis in vivoObjective In vivo transfection rate of shRNA lentivirus in mouse heart via vena dosalis penis injection was evaluated and the inhibiting effects of lentivirus mediated RNA interference on al,3GT and RelA were observed.Methods Vena dosalis penis injection was performed according to the following grouping:Group 1 normal saline control group, Group 2 negative control group, Group 3α1,3GTi-3-shRNA interfere group, Group 4 RelAi-3-shRNA interfere group, Group 5α1,3GTi-3-shRNA and RelAi-3-shRNA interfere group. Heart, liver and spleen were collected 140 hours after injection, and transfection rate and distribution of lentiviral vectors in different organs were evaluated by observing the EGFP expression of frozen slides of different organ under fluorescence microscope. After in vivo transfection, al,3GT and RelA mRNA gene expression, Galal,3Gal antigen expression, and RelA protein expression were observed by QPCR, immunofluorescence, and Westernblot respectively.Results Heart tissue of lentivirus interfered group showed a much stronger EGFP expression when compare with the normal saline group, especially in the vessel wall of the myocardium. There were also expression of EGFP in the liver and spleen tissues, but were much weaker than the heart tissue. There were significant decrease in target gene mRNA expressions in shRNA lentivirus interfere groups (p<0.01), but when compare between groups 3 or 4 and group 5, there were no significant difference (p>0.05). There was significant decrease in Galal,3Gal antigen expression in the blood vessel endothelium of the heart for al,3GTi-3-shRNA group when comparing with the control groups. There were significant decrease in RelA protein expression in groups 4 and 5 when comparing with control groups (p<0.01), but no significant difference when comparing between groups 4 and 5 (p>0.05).Conclusion High transfection rate in the heart is achieved by injection of lentiviral vectors via vena dorsalis penis. Suppression of al,3GT and RelA gene expressions are achieved by using the shRNA lentiviral vectors constructed in vivo. At the same time, expression of Galal,3Gal antigen and RelA protein are decreased too. The constructed lentiviral vectors can be used in xenotransplantation experiments.Part III Research on the Effects of RNA interference on al,3GT and RelA gene in cardiac xenotransplantation modelObjective The effects of shRNA lentiviral vector injection via vena dorsalis penis against rejection in mouse-to-rat heterotopic cardiac xenotransplantation model were observed, and the mechanism were approached.Methods Heterotopic cardiac xenotransplantation model was constructed by using the heart of BALB/c mouse 140 hours after lentiviral vector injection via vena dorsalis penis which was transplanted to the cervical part of F334 Rat. Heterotopic cardiac xenotransplantation model was constructed according to the following grouping,8 per group:Group 1 normal saline control group, Group 2 negative control group, Group 3α1,3GTi-3-shRNA interfere group, Group 4 RelAi-3-shRNA interfere group, Group 5α1,3GTi-3-shRNA and RelAi-3-shRNA interfere group. Survival time of the transplanted heart was observed, and the heart was collected when stopping beat, al, 3GT and RelA mRNA gene expression, Galal,3Gal antigen expression, and RelA protein expression were observed by QPCR, immunofluorescence, and Westernblot respectively.Results The survival time of donor heart in group 3 and 5 were longer than those in group 4 and control group with significant difference (p<0.01), however no significant Results The survival time of donor heart in group 3 and 5 were longer than those in group 4 and control group with significant difference (p<0.01), however no significant difference could be found between the above two groups respectively(p>0.05). The survival time between groups 4 and control group had no significant difference (p>0.05). The expression of the target genes before transplantion and after rejection within each shRNA interfered groups showed no significant difference (p>0.05), as same as the protein expression (p>0.05). However, there were increasing in expression of RelA protein in the rejected heart of the control group (p<0.01). And for the rejected heart in shRNA lentivirus interfered groups, the protein expressions of the target genes were still low when comparing with the control group (p<0.01).Conclusion The expression of al,3GT mRNA and Galal,3Gal antigen in the donor heart effected by al,3GTi-3-shRNA lentivirus vector are decreased showing that HAR is suppressed partly. As a result, this indicates that Galal,3 Gal antigen has contributed to the HAR in mouse-to-rat xenogeneic cardiac transplantation, but not the only xeno-antigen participated. RelAi-3-shRNA lentivirus can also reduce the expression of RelA mRNA and protein, but the survival period is still within HAR and the change of RelA protein expression does not prolong the survival time. |
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