| Background and ObjectiveABO incompatible(ABOi)kidney transplant(KT)is vast used in clinic as an effective method for the therapy of middle-advanced nephropathy.But even the ABOi-KT recipient received plasma treatment at preoperative and postoperative,the specific reaction between blood group antibody and blood group antigen of renal tissue cells could be the reason induced injury and dysfunction for kidney graft.And this still is the mainly barrier for ABOi-KT.Therefore,the simulating environment of ABO incompatible was established to explore the influence of blood group B antigen,which was expressed on human renal cell line HK2,for the activation and antibody production of B cells in this study,and further researches were aimed to the protein and action mechanism related to the B cells activation.The results will be presented the theoretical and experimental basis for the immune responses which were induced by blood group antigen of kidney tissue cells.Methods1.The influence of HK2 cells carried blood group B antigen for B cells activation from human blood group A(1)The type of blood group antigen on HK2 cells were detected by immunohistochemistry;(2)The peripheral blood mononuclear cells(PBMC)were isolated and divided into three groups: HK2 group,PBMC group and HK2+PBMC group;(3)The change of cellular morphology and quantity of HK2 cells were observed by optical microscope at days 2,4,8;(4)The phenotypes of T cells(CD3+,CD3+CD4+ and CD3+CD8+)and B cells(CD19+ and CD138+)were analyzed by flow cytometry(FCM)in PBMC and HK2+PBMC groups;(5)After extracted genome DNA from PBMC of PBMC and HK2+PBMC groups,the frequency and type of bases mutation in somatic hypermutation of class switch of Ig HM(Smu-SHM)were analyzed by sequencing.2.The influence of HK2 cells for the production of ABO blood group antibody(1)The concentration of blood group B antibody,Ig G and Ig M were analyzed by Dot-ELISA at days 2,4,8;(2)The concentration of blood group B antibody,Ig G and Ig M were analyzed by immunoturbidimetry at days 2,4,8;(3)The concentration of Ig G1,Ig G2,Ig G3 and Ig G4 were detected by ELISA at day 4.3.The identification and action mechanism of the related activation protein haptoglobin in B cells(1)The role of haptoglobin(HP),which had been identified as a related-activity protein of lymphocytes in our previous research,was analyzed by Gene Ontology(GO);(2)The lymphocytes(T cells and B cells)expressed HP were analyzed by flow cytometry(FCM);(3)The concentration of HP in HK2,PBMC and HK2+PBMC groups were analyzed by Dot-ELISA and immunoturbidimetry methods;(4)The proteins interacted with HP in 26 databases included Bio Grid,APID Interactomes and BAR were searched by Cytoscape software,and the function of proteins were analyzed by Kyoto Encyclopedia of Genes and Genomes Pathway(KEGG)and National Center for Biotechnology Information(NCBI);(5)The protein interacted with HP was identified and analyzed by Dot-ELISA.Results1.The influence of HK2 cells carried blood group B antigen for B cells activation of human blood group A(1)The HK2 cell line was identified to express human blood group B antigen;(2)Optical microscope observation showed that,with the time of culture,the HK2 cells were quickly proliferated in HK2 group and substantial declined in HK2+PBMC group;(3)FCM analysis showed that,after stimulated with HK2 cells,the T cell phenotype(CD3+?CD3+CD4+?CD3+CD8+)had no significant change,the CD19+ and CD138+ B cells were respectively significantly enhanced 3.07 fold(P<0.05)and 3.02 folds(P<0.05)at day 4;(4)Sequencing analysis appeared that the bases mutation frequency of Smu-SHM in HK2+PBMC group was significantly enhanced 8 fold and the mutation sites mainly distributed at 5' and 3' terminal.In addition,the GC target mutation,GC transition mutation and AT transition mutation were 55.56 %,70.00 % and 50.00 %,respectively.2.The influence of HK2 cells for the production of ABO blood group antibody(1)Dot-ELISA showed that,compared with PBMC group,the concentration of blood group B antibody was the highest at day 4 and significantly increased 5.28 fold(P<0.05)in HK2+PBMC group;the concentration of Ig G was increased with time and significantly increased 1.63 fold(P<0.05)at day 4;the concentration of Ig M had no significant change.(2)Immunoturbidimetry assay showed that,compared with PBMC group,the concentration of blood group B antibody was the highest at day 4 and significantly increased 2.35 fold(P<0.05)in HK2+PBMC group;the concentration of Ig G was significantly increased 3.6 and 2.81 folds at days 4 and 8(P<0.05),respectively;the concentration of Ig M had no significant change.Statistical analysis showed,the concentration of blood group B antibody in HK2+PBMC group was 44.00 % of the total concentration of Ig G and Ig M at day 4.(3)ELISA assay appeared that,compared with PBMC group,the concentration of Ig G1,Ig G2 and Ig G4 were respectively increased 1.89(P<0.01),1.30(P<0.05)and 1.16 fold(P<0.05)at day 4.3.The identification and action mechanism of the related activation protein haptoglobin in B cells(1)Base on our previous results which HP was an activation protein of human peripheral lymphocytes cultured in vitro,GO analysis found that HP may be involved in the regulation process of lymphocytes activation via costimulation pathway.(2)Flow cytometry assay showed that the HP was detected in B cells(CD19+)but not in T cells(CD3+).(3)Dot-ELISA and immunoturbidimetry analysis appeared that HP was detected out in PBMC protein,and the concentration in HK2+PBMC group(0.069 mg/ml)was significantly higher than PB group(0.052 mg/ml)to 1.34 fold(P<0.01).(4)Cytoscape software and KEGG analysis showed that there were 23 proteins could interacted with HP.And one of the protein Smad3,which was belonged to Mothers against decapentaplegic homolog family(SMAD),was a candidate protein to combine HP.(5)Dot-ELISA assay showed that the complexes HP-Smad3 and HP-Phospho-Smad3 were detected out in PBMC protein.Conclusion1.The B cells activation and Ig class switch recombination were induced by HK2 cells which carried blood group B antigen,and the mainly antibody produced by B cells was Ig G;the blood group B antigen was an important dominant antigen in HK2 cells in induced B cells activation.2.Haptoglobin(HP),a protein associated to B cells activation,can interact with Smad3 and phosphorylated Smad3,and may be to influence the B cells activation and Ig class switch recombination via Smad3 signal pathway. |
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