| OBJECTIVE: To explore the therapic effects of Tα146-162-iMDCs in C57BL/6 mice with EAMG. To illustrate whether this therapiceffect was related with the change of B cell activation, the regulationeffect of Cbl and Cbl-b on BCR signaling were investigated, andanti-AchR Abs isotypes were observed dynamicly.METHODS: (1) 34 adult male C57BL/6 mice were randomlydivided into EAMG groups(A group, n=12), the prevention group(Bgroup, n=12) and control group(C group, n=10); (2) Dendritic precursorcells from mice bone marrow cultured and differentiated in mediumsupplemented with GM-CSF and IL-10. iMDCs were collected at day 6and pulsed with Tα146-162 before use. Cell surface markers of DC wereanalysed by flow cytometry; (3) Mice of A group and B group wereevaluated for clinical score till the day they were put to death. (4) On theday 0, and 15, 45, 75 after the first immunization, anti-AchR Abs IgM,IgG,IgG1,IgG2b and IgG2c were measured by ELISA. (5) The mRNAexpression of Cbl and Cbl-b were detected on the day 90 after the termination of experiment by RT-PCR. (6) The expression andphosphorylation of Syk, Lyn, Btk and PLC-γ2 protein were measured byWestern Blot.RESULTS: (1) 9/12 (75％) mice of A group and 3/12 (25％) miceof B group were developed the accumulated incidence, the differencewas significant (p＜0.05). The average clinical score of A group and Bgroup were 1.69±1.12vs0.35±0.67(p＜0.01) at the termination ofexperiment. There was no mouse of C group developed the accumulatedincidence. (2) There was no significant difference of the level of IgMbetween A and B groups. After the first immunization, the level of IgMof A and B groups were higher than it of C group (p＜0.01); After thefirst immunization, the level of IgG and IgG1 of A and B groups werehigher than those of C group (p＜0.01). Compared with A group, the levelof IgG and IgG1 orB group were lower, on the day 15, p＜0.05, on theday 45 and 75, p＜0.01; After the first immunization, the level of IgG2bof A and B groups were higher than it of C group (p＜0.01). Comparedwith A group, after the second immunization, the level of IgG2b of Bgroup was lower, on the day 45, p＜0.05, on the day 75, p＜0.01; After thefirst immunization, the level of IgG2c of A and B groups were higherthan those of C group (p＜0.01). But there was no significant differenceof the level of IgG2c between A and B groups. (3) The mRNAexpression of Cbl and Cbl-b in spleen and lymphonode of the mice of A group were lower than those of C group (p＜0.01). Compared with Agroup, the mRNA expression of Cb1 and Cb1-b of B groups were higher(p＜0.05), but still lower than those of C group (p＜0.05); (4) Theexpression and phosphorylation of Syk and PLC-γ2 protein in spleenand lymphonode of the mice of A group were higher than those of Cgroup (p＜0.01) and those of B group (p＜0.05). Compared with C group,those of B group were higher (p＜0.05); The expression andphosphorylation of Lyn protein in spleen and lymphonode of the mice ofA (p＜0.01) and B (p＜0.05) groups were lower than those of C group.Compared with A group, those of B group were higher (p＜0.05); Theexpression of Btk protein in spleen and lymphonode of the mice of A(p＜0.01) and B (p＜0.05) groups were higher than those of C group. Andthose of B groups were lower than those of A group (p＜0.05). But therewere no remarkable differences among the phosphorylation of Btkprotein of three groups.CONCLUSION:1. Tα1 146-162-iMDCs can prevent EAMG.2. The pathogenesis of EAMG is closely correlated with B cellactivation induced by the negative regulation of Cb1 and Cb1-b on BCRsignaling.3. Tα146-162-iMDCs probably induce tolerance by enhancing thenegative regulation of Cb1 and Cb1-b on BCR signaling. 4. Tα146-162-iMDCs can probably ameliorate EAMG by affectingthe leval of IgG1 and IgG2b. |
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