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小鼠GITRL蛋白对Th17细胞的影响及其在小鼠CIA发病中的作用 |
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ontrol protein was injected into the CIA mice,and its role on the development of the disease(clinical scores,CIA incidence,histology) was observed.The frequency and number of Th17 cells from spleens and draining lymph nodes,and the expression of transcription factor RORγt were analyzed by FCM;the levels of IL-17 in serum were examined by ELISA method.Results:(1) The mGITRL fusion protein was expressed in E.coli BL-21 at 30℃after 1mmol/L IPTG induction for 6 hours.The molecular weight of the fusion protein was about 40KD and the purity was larger than 90%. Rabbit anti-mouse GITRL polyclonal antibody was prepared.It showed that the titer of the antibody was 1:20000 by ELISA and the mGITRL protein could be specifically recognized by the antibody by Western blot.(2) The purity of murine CD4+T cells purified by MACS is larger than 95%.Compared with the control protein(14.1%),we observed an increase frequency of Th17 cells(16.1%) in the culture supplemented with mGITRL protein.An increase level of IL-17 in the supematant was systematically along with increasing concentration of mGITRL protein (0.5、1、5、10μg/ml) from 387.51 pg/ml to 512.70pg/ml and the frequency of Th17 cells was from 15.81%to 17.90%.(3) Compared with the control group(CTL group),the frequency of Th17 cells in the murine spleens of GITRL group increased significantly (0.86±0.40%vs 1.77±0.49%,p＜0.01) and the percentage of CD4+RORγt+T cell also increased significantly(1.21±0.55%vs 1.91±0.60%,p＜0.05),and the level of IL-17 in murine serum of GITRL group (3.68±1.37 pg/ml vs 6.54±1.32 pg/ml,p＜0.05) increased significantly.(4) The model of CIA was established successfully.With the CIA model,we observed that compared with the the occurrence time(day 32) and the peak time(day 46) of CTL-CIA group,the group dealed with mGITRL protein(GITRL-CIA group) had an earlier onset of symptoms (day 28) and reached the peak score rapidly(day 38).The peak score of CTL-CIA group is 9.1,and the GITRL-CIA group reached 10.1.The pat 上一页 [1] [2] [3] [4] [5] 下一页
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