Y Chromosome Traces The Micro-chimerism Of Exogenous Tolerant DCs In CIA Rats

Objective:Detecting the sex-determining region of Y-chromosome（SRY）gene by using small animal live imaging technology,fluorescent tracer and polymerase chain reaction（PCR）technology,to search for input collagen-induced joints The fate and distribution of tolerogenic dendritic cells（tolerogenic dendritic cells,tol DC）in collagen-induced arthritis（CIA）rats,to explore the formation of micro-chimerism of exogenous tol DC in CIA rats Evidence,and preliminary exploration of the immune sentinel molecule programmed cell death-1（programmed cell death-1,PD-1）/programmed cell death-ligand 1（programmed cell death-1）in the local microenvironment of CIA rat joints.The expression of ligand 1,PD-L1),and then to explore the intervention mechanism of tol DC in CIA rats.Methods:1.Preparation of male Sprague Dawley（SD）rat bone marrow-derived tol DC:（1）bone marrow-derived mononuclear cells were isolated from the tibia of normal male SD rat.Granulocyte-macrophage colony stimulating factor（GM-CSF）and interleukin-4（IL-4）were used to induce monocytes into DCs,and nuclear factor-kappa B oligodeoxynucleotide decoy（NF-κB ODN Decoy）was added with cytokines to transform DCs into tol DCs.（2）According to the different processing methods of culturing bone marrow-derived mononuclear cells,cell experiment was divided into four groups:Control-DC group,Decoy-DC group,lipopolysaccharide（LPS）-DC group and LPS-Decoy-DC group.The cells at different stages and the cells stained by Giemsa were observed under the inverted microscope,and the number of viable cells was evaluated by trypan blue staining.Flow cytometry was used to detect the surface specific marker of rat DC（OX62）and maturity-related molecules（CD80and CD86）,Cell Counting Kit-8（CCK-8）was used to detect the proliferation of spleen-derived lymphocytes in male Wistar rats stimulated by DCs in each group.2.To find evidence that the infused male tol DC formed micro-chimerism in female CIA rats:（1）Preparation of BIIC-Decoy-DC^?suspension:NF-κB ODN Decoy induced rat tol DC loaded with bovineⅡcollagen（BⅡC）is BⅡC-Decoy-DC.BⅡC-Decoy-DC stained with 1,1’-dioctadecyl-3,3,3’,’-tetrame-thylindotricarbocyan-ine iodide（Di R）cell membrane staining solution is BIIC-Decoy-DC^?.（2）The CIA model was established by intradermal injection of BIIC emulsion into the left foot of female SD rats,and the experiment was set as a CIA model group:Equal volume of PBS was injected into the tail vein on the 5th day of modeling;BIIC-Decoy-DC^?treatment group:About 5×10⁶BIIC-Decoy-DC^?suspension was injected into the tail vein on the 5th day of modeling.（3）In vivo imaging system（IVIS）analysis:the above-prepared BIIC-Decoy-DC^?suspension was injected into CIA rats,at 4h,24h,5d,10d,15d,IVIS Continuously observe the fluorescence localization and intensity in living rats;on the 15th day of cell infusion,the CIA rats were anesthetized and sacrificed,and the heart,liver,spleen,lung,kidney and mesenteric lymph nodes were placed under IVIS for fluorescence observation.And freezed them for later use.The left hind ankle joint of the rat was taken to make pathological sections,and the histopathological changes were observed.（4）PCR detection of SRY gene from male rats in the BIIC-Decoy-DC^?treatment group and CIA model group rat cryopreserved organs（heart,liver,spleen,lung,kidney,mesenteric lymph nodes）to confirm whether the micro-chimerism is from BIIC-Decoy-DC^?.3.Changes in the sentinel molecule PD-1/PD-L1 of the joint immune microenvironment:Immunohistochemical technique was used to detect the expression of PD-1/PD-L1 in the local microenvironment of the left hind ankle of rats in the BIIC-Decoy-DC^?treatment group and the CIA model group.Results:（1）Performance evaluation of tol DC prepared by NF-κB ODN Decoy technology:Compared with the Control-DC group,the Decoy-DC group cell colonies were smaller and fewer under the inverted microscope,and Giemsa staining showed that the Decoy-DC group had more surface protrusions.The Control-DC group was short and few;trypan blue staining showed that the cell viability of the Decoy-DC group was>93%;flow cytometry showed that the OX-62 expression of DCs in the Control-DC group and the Decoy-DC group was>85%.However,the expression of DC CD80 and CD86 in the Decoy-DC group was lower than that of the Control-DC group（P<0.05）;CCK-8 results showed that the optical density(OD₄₅₀)value of the cells in the Decoy-DC group at 450 nm was lower than that of the Control-DC group.In the DC group（P<0.05）,OD₄₅₀was up-regulated after LPS treatment,and OD₄₅₀in the LPS-Decoy-DC group was lower than that in the LPS-DC group（P<0.05）.（2）The pathological results of the ankle joint showed that compared with the CIA model group,the pathological performance of the ankle joint in the BIIC-Decoy-DC^?treatment group was significantly improved.（3）IVIS analysis results:After BIIC-Decoy-DC^?was injected into the rat body,it could migrate with the blood to the tissues and organs of the rat’s whole body,and existed in many parts,especially in the chest and abdomen and the left ankle joint of the rat Which showed Strong fluorescence signal,.The fluorescence signal decreased with the increase of time.The fluorescence signals of the isolated organs were stronger in the liver,lung,spleen and mesenteric lymph nodes,and the fluorescence signals of the heart and kidney were weak.This result is basically consistent with the fluorescence expression observed by the research group using splenic-derived BIIC-Decoy-DC^?in our early study.（4）PCR test results showed that in the BIIC-Decoy-DC^?treatment group,the specific SRY gene fragment of the bone marrow-derived BIIC-Decoy-DC^?of the donor male rat was detected in the liver,lung,spleen and mesenteric lymph nodes of female CIA rats on the 15th day after infusion of bone marrow-derived BIIC-Decoy-DC^?in male rats.The PCR amplified product was subjected to agarosegel electrophoresis,and the target band was positive,but SRY was not detected in the heart and kidney.（5）The results of immunohistochemical technique showed that:Compared with the CIA model group,the average optical density of PD-1/PD-L1 in the left ankle joint of rats in the BIIC-Decoy-DC^?treatment group increased（P<0.05）.Conclusions:（1）Tol DC prepared by NF-κB ODN Decoy technology can effectively intervene in the further development of joint inflammation and pathological changes in CIA rats.（2）Bone marrow-derived BIIC-Decoy-DC（?）can form micro-chimerism in CIA rats.（3）PCR technology detected the SRY gene in the lung,liver,spleen and mesenteric lymph nodes,which was basically consistent with the results obtained by in vivo fluorescence imaging technology,and mutually confirmed the existence of micro-chimerism.（4）Small animal live imaging technology and PCR technology have their own advantages and differences,and the two can be complementary to each other or used depending on the conditions.（5）Chimeric tol DC may regulate the immune microenvironment by up-regulating PD-1/PD-L1 in the joints,thereby reducing autoimmune damage in CIA rats.