小鼠GITRL蛋白对Th17细胞的影响及其在小鼠CIA发病中的作用

【Abstract】 Objective:Glucocorticoid-induced tumor necrosis factor receptor(GITR) and its ligand(GITRL) play significant roles in regulating immune responses. To explore the role of mGITRL on murine Th17 cells in vitro and in vivo; and to observe its role on the development of the collagen-induced arthritis(CIA) in mice and research the mechanism.Methods:(1) The murine GITRL fusion protein was expressed in E.coli BL-21 with Isopropylβ-D-1- thiogalacto- pyranoside(IPTG),and identified by SDS-PAGE.The target protein was purified by IMAC column,and then identified by SDS-PAGE.The endotoxin was removed by Endotoxin Removal Kit.The rabbit was immunized by the purified recombinant mGITRL protein as antigen combined with the Freud s complete or incomplete adjuvant for three times.The rabbit anti-mouse GITRL serum was collected,the titer and specificity of the antibody was detected by ELISA or Western blot respectively.(2) CD4+T cells were purified by MACS from the normal murine spleens,and the purity of...更多 the cells was analyzed by flow cytometry (FCM).The recombinant mGITRL protein or the control protein was added into the CD4+T cells culture,and incubated under a Th17-differentiation condition for 96 hours.The frequency of Th17 cells was analyzed by FCM,and the supernatant was harvested to dectect IL-17 by ELISA method.(3) The mGITRL protein or the control protein was injected into normal mice,respectively.After 10 days,the frequency of Th17 cells and the expression of transcription factor RORγt from spleens were measured by FCM,the IL-17 in serum were examined by ELISA method.(4) The collagen-induced arthritis(CIA) model in DBA/1 mice was established.The mGITRL protein or the c

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