Adoptive Immune Tolerance By Recipient Murine Semi-mature Dendritic Cells Loaded With Trophoblastic Cells Antigen

Part I Cultivation and identify of murine myeloid semi-mature dendritic cellsObjective To induce mouse bone marrow cells into semi-mature dendritic cells in vitro,and study the phenotype and function of semi-mature dendritic cells.Methods The progenitors of dendritic cells in bone marrow were cultured in the presence of granulocyte macrophage colony stimulating factor(GM-CSF) in vitro,then small interfer RNA(siRNA) of MyD88 was addd at day 7,and LPS was added at day 9,the phenotype and regulatory capacity of the cells harvested were analyzed by FASC ,ELISA and primary and secondary mixed lymphocyte reaction.Results the harvested cells were semi-mature dendritic cells whose phenotype was CD11+CD40midCD80lowCD86lowMHC-IImid. semi-mature DCs produce high IL-10 and low IL-12 which led to a distinct Th2 cytokine profile . In primary MLR,semi-mature DCs could not stimulate the proliferation of T cells. In secondary MLR,After infusion of donor semi-mature DCs , responder cells isolated from the recipient mice showed donor-specific hyporesponsiveness to restimulation by donor cells.Conclusions Immature DCs could be induced by GM-CSF and siRNA of MyD88 in vitro, then become semi-mature DCs after stimulation by LPS. These semi- mature DCs are more tolerogenic than immature DCs.Part II Effects of loading trophoblastic cells antigen on phenotype and function of semi-mature dendritic cells Experiment One The culture and indentify of trophoblastic cellsObjective To cultivate and identify murine trophoblastic cells in vitro and analyze its purity.Methods Murine trophoblastic cells obtained by culture of ectoplacenta cone (EPC) tissue in vitro was identified by immunohistochemistry stain and its purity was analyzed by FACS.Results Quantity sufficient trophoblastic cells was obtained . the result of immunohistochemistry stain show PLP-B positive and the purity was more than 90% analyzed by FACS.Conclusions the purity and quantity of trophoblastic cells were satisfying for next experiment.Experiment two The phonetype and function of semi-mature DCs loaded with trophoblastic cells antigen.Objective To study the phenotype and function of semi-mature dendritic cells after loaded with trophoblastic cells antigen .Methods Semi-mature DCs were loaded with spleen cells and trophoblastic cells antigen. phenotype was analyzed by FACS , autocrine cytokine IL10 and IL12 was detected by ELISA. the function of stimulating T cells proliferation was evalued by MLR.Results Expression of MHC-II and costimulatory molecules CD86 were elevated after loaded with spleen cell antigen ,while expression of costimulatory molecules CD40 and CD80 was not changed . semi-mature DCs produce a little more IL-2 (P>0.05 no significant deviation) after loaded with trophoblastic cells antigen,MHC-II expression was elevated (P<0.05) , but costimulatory molecules expression was not changed ,IL-10/IL-12 ratio produced by semi-mature DCs increased .semi-mature DCs of both groups could not stimulate T cells proliferation effectively.Conclusions After loaded with trophoblastic cells antigen ,costimulatory molecules expression of semi-mature DCs do not change . the semi-mature DCs show a more distinct Th2 cytokine profile characterized and are tolerogenic DCs. Part III Effects of using recipient semi-mature dendritic cells pulsed with trophoblastic cells antigen on murine cardiac allograft survival time Experiment one Improved technique of cervical heterotopic heart transplantation in miceObjective To improve the anastomosis technique of mouse cervical heterotopic heart transplantation and establish a moderate -difficulty methods having long-term paterncy rate .Methods By using Hybrid method, the donor heart innominate artery and the right common carotid artery of the recipient were anastomosed using the end-to-end suture technique while the pulmonary artery of the graft was anastomosed end to end with external jugular vein of recipient by a 24G cuff. To compare the instant result of operation and survive time of graft heart of three groups ( Hybrid,both- suture and both-cuff method).Results The achievement radio of operation using Hybrid methods was good ; the difficulty and operating time was between both-cuff and both-suture method ,but Hybrid methods had better long-term paterncy rate of artery anastomose than both-cuff method which had no significant difference with both-suture method. there was no significant difference in survive time among three groups.Conclusions Hybrid method is a satisfying technique of cervical heterotopic heart transplantation in mice.Experiment two Marked prolongation of murine cardiac allograft survival using recipient semi-mature dendritic cells pulsed with trophoblastic cells antigenObjective To investigate the effect of recipient semi-mature dendritic cells pulsed with trophoblastic cells antigen on inducing murine cardiac allograft tolerance.Methods the cervical heterotopic heart transplantation was performed from C57BL/6 to BALB/c mice after recipients were given one injection of recipient semi-mature dendritic cells pulsed with trophoblastic cells antigen through the tail vein at 7 days before operation. Survival time of renalallografts was observed and splenic cell reaction of the recipient s to donors antigens were assayed by MLR.Results Injection of recipient semi-mature DCs loaded with trophoblastic cells antigen through the tail vein at 7 days before the heart transplantation significantly prolonged heart allograft survive time. The T cells in the recipient spleen express higher IL-10 mRNA level and show immunological unresponsiveness to donor antigen.Conclusions recipient semi-mature DCs loaded with trophoblastic cells antigen can prolong the survive time of cardiacal allograft and induce transplant tolerance.