Study Of Inducing Tolerogenic Dendritic Cells In Vitro And Immunologic Tolerance In Vivo

Objective:To establish the culture method of tolerogenic dendritic cells(tDCs) in vitro,followed by identification of its morphology,immunological phenotype and metergasis,In the mouse established the method of tDCs to induce specific immunological tolerance in vivo. In order to supply the experiment mechanism to tDCs in the prevent of transplantation rejection and the therapy of autoimmune disease.Methods:Male Balb/c mice are used as donor and female Kunming mice as receptor.Tolerogenic dendritic cells were generated from bone marrow precursors cultured with GM-CSF,IL-4,VIP,DXM,LPS. The cells were divied into five groups: blank group(not add any angent),common-DC group(GM-CSF+IL-4),VIP-DC group(GM-CSF+VIP),DXM-DCgroup(GM-CSF+DXM),VIP+DXM–DC group (GM-CSF+VIP+DXM)。The cell morphological differences were examined under optical microscope. The differences on cell phenotype were analyzed by flow cytometry(FCM). The capability to stimulate the proliferation of lymphocytes were examined through MTT method. IL-10 and IL-12 of the culture medium were measured with enzyme linked immunosorbent assay (ELISA). The animal transfusion experiment were divied into four groups: Blank group(not with any treatment),bone marrow group(transfusion bone marrow),common-DC group(transfusion common-DCs),tolerogenic-DC group(transfusion tolerogenic-DCs) . Three days after transplantation transplant the pieces of donor's spleen to receptor. Twenty days after transplantation take the pieces of transplanted spleen into pathological section. A month after transplantation, to detect CD80,CD86,CD40,CD11c and CD4+CD25+of spleen single caryocells of receptor through FCM.Using PKH26 to mark tDCs for site-specific study:A mouth after transplantation, Take the liver,spleen,lymphnode of recptor transfused with PKH26 marked tDCs into stamp pieces and froze section, then examined under fluorescence microscope.Results:The cells possessed morphological characteristic of typical DCs. Compared to the common-DC group,The VIP-DC group,DXM-DC group and VIP+DXM–DC group lowly express CD80,CD86,CD40 and CD11c, induce light lymphocytes proliferation,the increase secretion of IL-10,and the decrease of secretion of IL-12 in the culture medium.(P<0.05) The VIP(40ng/ml)+DXM(10ng/ml) group is most different to other groups. (P<0.05) In the transfusion experiment of mouse, the tDCs group present grayish red, and establish blood supply to surrounding structure; in the histopathologic examination it didn't see obviously denaturation and necrosis.But the Blank group,bone marrow group,common-DC group present grayish white,light grayish red,grayish brown, and didn't establish blood supply to surrounding structure; in the histopathologic examination it see obviously necrosis. The CD4+CD25+ cell of tDCs group is 18.15±0.66%,and the normal mouse is 6.5±0.55%.(P<0.05) Compared to the common-DC group,the tDCs group lowly express CD80,CD86,CD40 and CD11c. (P<0.05) Using PKH26 to mark tDCs, In the stamp pieces of mouse transfused with PKH26 marked tDCs, the quantity of fluorescent cells in spleen of mouse(44±3/HP) is at most,liver (26±2/HP),lymphnode(3±1/HP). In the frozen section of mouse transfused with PKH26 marked tDCs, the quantity of fluorescent cells in spleen of mouse(43±3/HP) is at most,liver (24±2/HP),lymphnode(2±1/HP).Conclusion:Tolerogenic dendritic cells were generated from bone marrow precursors cultured with GM-CSF,VIP/DXM,LPS. Compared to the common-DCs,The tDCs lowly express CD80,CD86,CD40 and CD11c, induce light lymphocytes proliferation,the increase secretion of IL-10,and the decrease of secretion of IL-12 in the culture medium.Among all the culture condition the recombinant of VIP40ng/ml and DXM10ng/ml is the best culture condition.Transfused tDCs to the mouse, The CD4+CD25+ cell of tDCs group is high,and lowly express CD80,CD86,CD40 and CD11c.Transfused tDCs in abdominal cavity, the fluorescent cells migrated to spleen,liver,and lymphnode, the quantity of fluorescent cells in spleen is at most . The safety of transfused tDCs in abdominal cavity is high, tDCs could form stabilized engomphosis to the receptor,and persistant more than one month.