Prolongation Of Rat Intestinal Recipients’ Survival By Administration Of Donor Myd88-silenced Immature Dendritic Cells

Chapter 1 Establishment of Intestinal Transplantation Model in RatsObjective To investigate the surgical procedures and to establish stable intestinal transplantation model in rats. Methods Segmental heterotopic intestinal transplantation was performed from donor F344 rats to recipient SD rats. A segment of intestine about 10cm with vessels was harvested from donor F344 rats. The vascular reconstruction was as follows: donor superior mesenteric artery was anastomosed to recipient infrarenal aorta and donor portal vein to recipient inferior vena cave. The proximal end of graft was ligatured and the distal end was exteriorized as a stoma on the abdominal wall.Results A total of 52 rats underwent intestinal transplantation with 43 rats survived more than 3 days. The donor operation time was 42.1±8.7 min, and the recipient’s 67.6±11.5 min. Of the operation time, the anatomosis of artery toke 10.5±3.7 min, and that of vein 12.7±3.6 min. The cold ischemia time of graft was 21.9±7.6 min and the warm ischemia time 23.9±3.1 min. The survival of recipients without any preconditioning varied from 4.5days to 10 days, with an average of 5.9±1.9 days.Conclusion This intestinal transplantation model by us is stable, reliable and useful for the further study of intestinal transplantation. Chapter 2 In vitro induction, proliferation and identification of immature dendritic cells derived from rat bone marrow cellsObjective To study the effective method for in vitro induction, proliferation and indentification of rat bone marrow-derived immature dendritic cells (iDC).Methods Bone marrow progenitor cells were obtained from F344 rat femoral and tibial bones and then cultured in the culture medium with 5ng/ml GM-CSF. On every other day, half of the medium was removed and equal volume of fresh medium was added. On day 6, non-adherent and loosely adherent cells were harvested and identified as iDC by flow cytometry, electronic microscope and MLR. Results On day 6 of culture, iDC were harvested and identified. Morphological analysis by electronic microscope showed the typical propery of iDC. Surface phenotype assay by flow cytometry indicated the low to moderate expression of MHC classⅡ, and low expression of CD80 and CD86 of iDC. MLR meant that iDC induced lower T-cell proliferation than mature DC.Conclusion It is an efficient and feasible method in this study of inducing and proliferating rat bone marrow-derived iDC in vitro. And this makes a foundation of the basic and clinical study of iDC.Chapter 3 Small interfering RNA induced MyD88 gene silence in iDCObjective To investigate whether chemically synthesized siRNA could be transfected into iDC efficiently by lipofectamine and could induce specific gene silence in iDC.Methods iDC were generated by culturing bone marrow progenitor cells of rats with GM-CSF in vitro. Chemically synthesized MyD88-siRNA at the concentration of 1μg/500μl was transfected into iDC by lipofectamine. The efficiency of transfection and the expression of MHC II and costimulatory molecules of MyD88-silenced iDC were analysed with flow cytometry. RT-PCR assay was used to detect MyD88 mRNA transcription and Western blot to detect the expression of MyD88 protein.Results MyD88-siRNA was transfected into iDC with high transfection efficiency. And the transfection procedures did not affect the immature phenotype property of iDC. MyD88 mRNA transcription and the expression of MyD88 protein of MyD88-silenced iDC were at a very low level.Conclusion Transfection of siRNA by lipofectamine is a practical and effective way to induce MyD88 gene silence.Chapter 4 Administration of MyD88-silenced iDC prolonged the recipients’survival of intestinal transplantation in ratsObjective To investigate whether administration of MyD88-silenced iDC could prolong the recipients’survival of intestinal transplantation in rats.Methods iDC from F344 rats were generated by culturing bone marrow progenitor cells of rats with GM-CSF in vitro and Chemically synthesized MyD88-siRNA was transfected into iDC by lipofectamine. Segmental heterotopic intestinal transplantation was performed from donor F344 rats to recipient SD rats. Recipient rats were divided into four groups according to the preconditionings that recipients received 7days before transplantation, i.e. Group A, in which recipients were injected with one milliliter Normal Saline; Group B, in which recipients were injected with iDC; Group C, in which recipients were injected with negative control siRNA- silenced iDC and Group D, in which recipients were injected with MyD88-silenced iDC. The survival of recipients and histologic examination of graft intestine was evaluated after intestinal transplantation. Results Survival time of recipients in Group A, B, C and D was 5.9±1.9, 11.0±1.87, 10.2±1.64, 18.6±3.60days, respectively. There was no statistical significance of recipient survival between Group B and C, but survival in Group B and C was longer than that in group A. Survival in Group D was significantly longer than that in other three groups. Histologic examination indicated that mucosa structural destruction and rejection of intestine grafts in Group D were remarkably alleviated compared with other three groups.Conclusion Administration of MyD88-silenced iDC could prolong the survival time of grafts and recipients and alleviate the graft rejection.Chapter 5 Effects of MyD88 gene silence on immune function of iDCObjective To investigate the effects of MyD88 gene silence on immune function of iDC and the mechanisms by which MyD88 silence enhances the tolerogenicity of iDC in intestinal transplantation in rats.Methods iDC from F344 rats were generated by culturing bone marrow progenitor cells of rats with GM-CSF in vitro. Chemically synthesized MyD88-siRNA was transfected into iDC by lipofectamine. Pro-inflammatory cytokines such as IL-12 and TNF-αby MyD88-silenced iDC when stimulated by LPS were determined with ELISA. Recipient spleens were harvested seven days after intestinal transplantation and T cells were isolated and purified. Then these T cells were stimulated by donor spleen lymphocytes inactivated by mitomycin C and cytokines such as IFN-γand IL-4 by T cells were determined with ELISA. Mixed lymphocyte reaction was adopted to determine the ability to stimulate T cell proliferation between MyD88-silenced iDC and wild type iDC. FAM-labeled MyD88-silenced iDC or negative control-silenced iDC was injected into SD rats and the distribution of these cells in lymphatic organs was assayed with fluorescence microscope.Results MyD88-silenced iDC produced less cytokines of IL-12 and TNF-αwhen compared with the wild type iDC. T cells from recipient spleens pre-treated with MyD88-silenced iDC produced more IL-4 but less IFN-γthan those pre-treated with wile type iDC or negative control-silenced iDC. There was no statistical significance of the ability to stimulate T cell proliferation between MyD88-silenced iDC and wild type iDC and there also no difference of distribution in lymphatic organs between MyD88-silenced iDC and negative control-silenced iDC.Conclusion Inability to produce pro-inflammatory cytokines, especially IL-12, and therefore inducing the Th2 polarization of Th0 cells may be one of the underlying mechanisms by which administration of MyD88-silenced iDC alleviated the immune rejection of intestine graft and prolonged the recipient survival.