Observation Of The Microchimerism Status Of Tolerant Dendritic Cells Infusion In Collagen Induced Arthritis Rats

Objective:To observe the microchimerism status of tolerant dendritic cells(DC)inputted into collagen induced arthritis(CIA)rats,and to investigate the mechanism of the rapeutic intervention of tolerant DC on CIA rats.Methods:CIA female SD rats and normal male SD rats were used as experimental animals.1.The construction of tolerant DC derived from CIA female SD rats and normal male SD rats induced by Nuclear factor-kappa B oligodeoxynucleotide decoy(NF-KB ODN Decoy):(1)The left metatarsal of normal female SD rats were injected with 200μl bovine type Ⅱ collagen(BIIC)emulsion for CIA model;(2)Spleen mononuclear cells from CIA female SD rats(13days after primary immunization)and normal male SD rats,Granulocyte-macrophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4)were cultured as DC,which were induced by NF-κB ODN Decoy as tolerant DC;(3)The group of culture DCs:the culture cells derived from CIA female SD rats and normal male SD rats were divided into 4 groups respectively;(4)The observation of biological traits of tolerant DC:Inverted microscopy and Giemsa staining were used to observe cells morphological characteristics,Trypan blue staining was uesd to count the cell viable rate,the expression of OX-62,a DC-specific marker,was determined by flow cytometry,the expression of CD80 and CD86 molecules on the surface of DC and the reaction of allogeneic mixed lymphocytes were evaluated the tolerance and stability of tolerant DC;2.The observation of microchimeric status of tolerant DC inputted into CIA rats:(1)The tolerant DC derived from CIA female SD rats and normal male SD rats loaded BIIC,namely BIIC-Decoy DC,labeled DiR fluorescent dye,namely BIIC-Decoy DC(?);(2)Trypan blue staining to evalute the cell viable rate before and after DiR fluorescent dye labeling,and to assess the activity of BIIC-Decoy DC(?)by MTT assay;(3)To observe the fluorescence intensity of BIIC-Decoy DC(?);(4)In vivo imaging experiment groups:group A:DiR fluorescent dye labeled the BIIC-Decoy DC(namely BIIC-Decoy DC(?))of derived from allogeneic normal male SD rats,in the course of 5days,caudal vein inputted in CIA rats(the course of early CIA);group B:BIIC-Decoy DC(?)derived from allogeneic normal male SD rats,in the course of 20th days,caudal vein inputted in CIA rats(the course of middle CIA);group C:BIIC-Decoy DC(?)derived from autologous CIA rats,caudal vein inputted in the course of middle CIA;Normal female control(NFc):BIIC-Decoy DC(?)derived from allogeneic normal male SD rats,caudal vein inputted in normal female SD rats;Normal male control(NMc);BIIC-Decoy DC(?)derived from autologous normal male SD rats,caudal vein inputted in normal male SD rats;Unlabeled cell control(cell control):unlabeled BⅡC-Decoy DC instead of labeled cells for group A,B,C cell control,recorded as group Acc,Bcc,Ccc.The model control(model control);Phosphate Buffered Saline(PBS)buffer instead of labeled cells as group A,B,C model control,recorded as group Amc,Bmc,Cmc.(5)In vivo imaging:dynamically observed migration,distribution and microchimerism status after the above two sources of BIIC-Decoy DC(?)were inputted into the rats at 4h,24h,5day,10day,20day,25day,etc;(6)Detection indexes after in vivo imaging:IVIS imaging of organs in vitro;serum was taken to detect the level of IL-2 and IFN-γ,IL-4 and IL-10;the ankle joint was taken for pathological section.Results:(1)The construction of tolerant DC:spleen mononuclear cells derived from CIA female and normal male SD rats were successfully induced to be tolerant DC,and the cell viable rate was>94%,and the cell purity was>85%;the expression of CD80 and CD86 on the surface of tolerant DCs and the results of allogeneic mixed lymphocyte reaction showed that the tolerance had a good stability.(2)The tolerant DC of derived from CIA female SD rats and normal male SD rats loaded BIIC,successfully constructed BIIC-Decoy DC,loaded DiR fluorescent dye,namely BIIC-Decoy DC(?);(3)Trypan blue staining:the cell viable rate of BIIC-Decoy DC before and after DiR fluorescent dye labeling were about 95%;(4)MTT assay:the results showed that the DiR fluorescent dye labeled BIIC-Decoy DC from the above two sources had no effect on cells activity;(5)The results of IVIS showed that BIIC-Decoy DC was successfully labeled with DiR dye and the fluorescence intensity increased with the increase of the number of cells;(6)In vivo imaging results of IVIS:the fluorescence signal was mainly concentrated in the chest and abdomen,and gradually migrated to other parts with the prolongation of imaging time,such as the joints of extremities,and the fluorescence intensity decreased gradually with the prolongation of imaging time,the fluorescence signal reached the peak at 24 h and was still visible until the 35th days;(7)IVIS results of in vivo organs imaging:the fluorescence signals are mainly concentrated in the lungs,spleen and lymph nodes,with the strongest fluorescent signal in the lungs and relatively weak in the liver,heart and kidney;(8)The detection of serum cytokine levels:in the early and middle course of the CIA rats model group,the expression levels of Thl cytokines such as IFN-γ、IL-2 were higher,while the expression levels of Th2 cytokines such as IL-10、IL-4 were lower,in the early and middle course of the CIA rats treatment group the expression of Th1 cytokines such as IFN-γ、IL-2 were significantly decreased and the expression of Th2 cytokines such as IL-10、IL-4 were significantly increased.(9)The results of ankle joint pathologicalithe BIIC-Decoy DC of derived from CIA female SD rats and normal male SD rats showed that good therapeutic effects in the early and middle course of the CIA rats;Conlusion:(1)The tolerant DC purity of both CIA female SD rats and normal male SD rats obtained from the modified DC extraction method was>85%;(2)The sources of CIA female SD rats and normal male SD rats BIIC-Decoy DC both had a good therapeutic effect on the course of early CIA rats and the course of middle CIA rats;(3)IVIS imaging dynamic observation of CIA female SD rats and normal male SD rats BIIC-Decoy DC caudal vein inputted in CIA rats,the fluorescence signal widely distributed in lung,spleen,lymph node and other tissues,it can still be detected until the 35th day,and the formation of microchimerism status can be preliminarily judged,meanwhile,the detection of serum cytokines levels showed that the pattern of cytokines in vivo shifted from Thl to Th2,which was beneficial to the formation of immune tolerance.