Prolongation Of Rat Intestinal Allograft Survival By Administration Of Donor IL-12 P35 Silenced Dendritic Cells

Chapter 1. Induction, proliferation and identification of dendritic cellsderived from bone marrow of rats in vitroObjective To study the effective method of isolation, culture andproliferation of rat bone marrow-derived dendritic cells (DCs) in vitro.Methods DCs were collected from bone marrow progenitor cells of ratscultured with GM-CSF, IL-4 and LPS in vitro. Morphology, expression ofspecial surface markers and functional abilities were observed.Results Morphological property of typical DC was observed in DCprogenitors at day 7 of culture. Positive special surface marker of MHCⅡ,OX62 and CD80 was observed by Flow cytometry analysis. The DC exhibitedactivities in stimulating the proliferation of allogenic T cells.Conclusion Modified Son's method is favorable in inducing andproliferating bone marrow-derived DC of rats in vitro. Chapter 2. Effects of IL-12 p35 silence on immune function of dendriticcells in ratsObjective To investigate the effects of IL-12 p35 silence on immunefunction of dendritic cells in rats.Methods DCs were generated by culturing bone marrow progenitor cellsof rats with GM-CSF, IL-4 and LPS in vitro. The IL-12 p35 siRNA wassynthesized and transfected into DCs. The expressions of CD80 and MHCⅡwere examined by flow cytometry. Reverse transcriptase PCR analysis wasused to detect IL-12 p35 mRNA transcription and ELISA was used to detectIL-12 and IL-10 protein levels, respectively. The costimulatory immunefunctions of DC was evaluated by mixed lymphocyte responses.Results IL-12 p35 siRNA silenced DC secreted much less IL-12, moreIL-10 and exhibited weak activity in stimulating the proliferation of allogenicT cells, but no changes on CD80 and MHCⅡexpressions.Conclusion IL-12 p35 siRNA interference exerts no effects on maturationof DC, but negative effect on immume function of DC. Chapter 3. IL-12 p35 silenced dendritic cells modulate immune responsesby blocking IL-12 signaling through JAK-STATpathway in T lymphocytesObjective To investigate whether IL-12 p35 silenced dendritic cellsmodulate immune responses by blocking IL-12 signaling through JAK-STATpathway in T lymphocytes.Methods A coculture of T cells and DCs was performed in Transwellsix-well plates. After 30 min coculture, T cells were harvested and STAT3,STAT4, JAK2 and TYK2 in T cells were analyzed by Western blot.Results IL-12 p35 silenced DC induced less tyrosine phosphorylation ofSTAT3, STAT4, JAK2 and TYK2 in T cells than those in untransfected DCConclusion IL-12 p35 silenced dendritic cells modulate immuneresponses by blocking IL-12 signaling through JAK-STAT pathway in Tlymphocytes. Chapter 4. Establishment of Small Bowel TransplantationModel in RatsObjective To investigate the surgical procedures and to establish stablesmall bowel transplantation (SBT) models in rats in order to study therejection of SBT.Methods Segmental heterotopic SBT was performed on SD rats. Thevascular reconstruction was to the infrarenal aorta and vena cave of therecipient. An segmental jejunectomy was performed in recipient. Theproximal jejunum of the graft was occluded, and a jejunostomy was createdby exteriorizing the distal end of the donor jejunum.Results The 5-days survival rate of 45 cases undergoing SBT after themodels stabilized was 87% (39/45). The donor operation took (60±10) min,and the recipient operation (90±15) min. The anatomosis of artery averaged(8±3) min, and the anatomoses of vein averaged (15±5) min.Conclusion This surgical technique is highly suitable for establishingstable small bowel transplantation (SBT) models in rats. Chapter 5. Study on prolonging intestinal allografts survival byadministration of IL-12 p35 silenced dendritic cellsObjective To investigate the effects of donor IL-12 p35 silenced dendriticcells (DCs) on prolonging the survival time of intestinal grafts in rats.Methods DCs from Wistar rats were collected from bone marrowprogenitor cells of donor rats cultured with GM-CSF and IL-4 in vitro. TheIL-12 p35 siRNA was chemically synthesized and transfected into DCs.Seven days before transplantation of small intestinal from donors, normalsaline (groupⅠ), non-silencing DCs (groupⅡ), IL-12 p35 silenced DCs (groupⅢ, groupⅣ, groupⅤ), were infused intraperitoneally into SD rats,respectively. CsA 8 mg/kg/d was infused intravenously in the recipient rats ingroupⅣpostoperatively, CsA 20 mg/kg/d was infused intravenously in therecipient rats in groupⅤpostoperatively. Small bowel transplantation wasperformed and the survival time of grafts was observed. Histologicexamination was evaluated, and serum IL-2 and INF-γlevels were assayed byELISA.Results The survival time of recipient grafts in groupⅢwas significantlylonger than that in groupⅠandⅡ. The degree of the inflammatory infiltratedcells and intestinal mucosa structural destruction were significantly milder ingroupⅢcompared with that in groupⅠandⅡ. The concentrations of recipientserum IL-2 and INF-γwere much lower in groupⅢcompared with those ingroupⅠandⅡ. The survival time of recipient grafts in groupⅣis longest, intestinal grafts mucosa structural destruction were mildset in groupⅣcompared with those in the other groups.Conclusion The preoperative treatment with IL-12 p35 silenced DCs caninhibit the rejection of intestinal allografts and prolong the grafts survival timeafter transplantation. When combined with small doses of CsApostoperatively, the recipient survival time could be prolonged further more.