The Anti-rejection Effect Of Blocking B7/CD28 Co-stimulatory Pathway By RNAi In Mice Small Bowel Transplantation

The research was performed using RNAi (RNA interference) technique for the modification of myeloid DC (Dendritic Cell) where the CD80 and CD86 expressions were inhibited to block B7/CD28 co-stimulatory pathway in order to study the mechanism of influence to T lymphocyte proliferation. The research is based on the protective effect induce by RNAi knocked down CD80 and CD86 genes expressions in donor derived DCs to allograft and anti-rejection mechanism in segmental small bowel allo-transplantation (SBT).Part â…  The research on the effect of RNAi to knock-down CD80 and CD86 expressions in vitroObjective To investigate the influence of RNAi on DC surface antigen CD80 and CD86 expressions and to explore the mechanism of influence to T lymphocyte proliferation after introducing DC modified by siRNA. Methods In vitro culture of the mice myeloid DC was performed by selective medium containing necessarycytokines for DC growth. The in vitro synthesis of SiRNA (small interference RNA) sequence for specified CD80 and CD86 mRNA and its transfection into the DC was performed respectively. The mRNA and surface antigen expression levels of CD80 and CD86 were assayed by semi-quantitative-RT-PCR and flowcytometry respectively with pre and post siRNA transfection. The influence on DC stimulating the proliferation ability of T lymphocyte resulting from silencing effect mediated by siRNA was observed through one-way MLC (Mixed Lymphocyte Culture). Results Through in vitro cell culture, about 1.5-2*107myeloid DCs can be harvested from each mouse. After the SiRNA transfection on DC, the mRNA levels of CD80 and CD86 was significantly lower accompanied by significantly decreased surface antigen expression levels from 85.85%, 98.31% to 32.58%, 34.69% respectively, but surface antigen for MHCII, CDllc were stable. The result of MLC demonstrated that the index of stimulation to T lymphocyte of DC knocked down by siRNA significantly decreased (P<0.0l), but the index level resumed after addition of IDO specific inhibitor 1-MT. Conclusion Large quantity of myeloid DC with typically histological configuration and high levels of MHCII+ and CDllc+ were able to obtain through in vitro culture in selective medium which activated naive T lymphocyte to generate immune response. Transfecting siRNA targeting to CD80 and CD86 mRNA with SiPort? Lipid into DC may efficiently and specifically inhibit CD80 and CD86 expression. The high expression of functional IDO can make stimulating ability of DC to allogenic T cells proliferation lower. Blocking B7/CD28 co-stimulatory pathway may cause high expression of functional IDO from DC.Part II The anti-rejection effect by blocking B7/CD28 co-stimulating pathway in small bowel transplantation ofmice.Objective To study the protective effect induce by RNAi knocked down CD80 and CD86 genes expressions in donor derived DCs to allograft and anti-rejection mechanism in segmental Small Bowel allo-transplantation. Methods Heterotopic small bowel transplantation (SBT) models were established with small mice by microsurgical technique. The experimental animals were divided as follows: allograft and isograft group being taken as the control, non-transfected DC group ( Intravenous infusion of donor derived non SiRNA transfected DCs (1 X 106) on 7th preoperative day), SiRNA transfected DC group ( Intravenous infusion of donor derived SiRNA transfected DCs (1 X 106) on 7th preoperative day). On the 7th post-operative day the graft survivals were individually havested and pathological evaluation was done. At the same time mRNA expression levels of IL-2, IFNy, IL-10 and IDO in the grafts' sample were determined by semi-quantitative-RT-PCR. Results Median Survival Time (MST) of the grafts in siRNA transfected DC group (MST 12 days) is significantly longer than allograft group (MST 6 days) (PO.01). Accompanied with significantly lower rejection pathological grading in comparison with allograft group and non transfected DC group (P<0.0\), but there was no significant differences seen in comparison between isograft groups (P>0.01). The lower mRNA expression levels of IL-2 and IFNy (PO.01) and higher level of IL10 and IDO (P<0.01) were observed in siRNA transfected DC group in comparison with allograft group and non-transfected DC group. Conclusion In mice heterotopicsmall bowel transplantation, donor-derived DCs in which molecules CD80 and CD86 have been knocked down through RNAi silencing could exhibit protective effect on grafts. This mechanism might be that specific T cell to antigen in recipients was inhibited. This inhibition effect seems to be relative with inducing IDO expression in graft and leading the deviation from Thl to Th2.