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目的探讨负载电鳗乙酰胆碱受体(TAChR)免疫优势肽段α146-162(Tα146-162)的不成熟髓源树突状细胞(iMDC),即Tα146-162-iMDC对实验性自身免疫性重症肌无力(EAMG)的预防作用及其机制。方法(1)24只C57BL/6小鼠随机分为Tα146-162-iMDC组、iMDC组、对照组、正常组,每组6只。Tα146-162-iMDC组、iMDC组和对照组小鼠皮下注射TAChR抗原乳剂(作为第0天)。第7天,Tα146-162-iMDC组皮下注射Tα146-162-iMDC,iMDC组和对照组分别注射对照iMDC和磷酸盐缓冲液(PBS)。正常组小鼠不做干预。第14天检测淋巴细胞增殖反应。(2)另外24只C57BL/6小鼠随机分为预防组和EAMG组。每只小鼠在第0、30、60天皮下注射抗原乳剂诱导EAMG。预防组每次免疫后3d皮下注射Tα146-162-iMDC,对照组皮下注射PBS。观察发病情况并进行肌力评分。第90天结束观察。(3)3H-TdR掺入法检测淋巴细胞增殖反应,酶联免疫吸附实验检测淋巴细胞培养液上清中干扰素(IFN)-γ、白细胞介素(IL)-6的含量。结果(1)Tα146-162-iMDC组、iMDC组、对照组和正常组TAChR特异性淋巴细胞增殖反应值(放射性荧光闪烁计数)分别为3314·1±571·3、7717·5±1283·4、8608·6±1458·9、922·9±109·3,与iMDC组和对照组相比,Tα146-162-iMDC组抗原特异性淋巴细胞增殖反应显著降低(P<0·01)。(2)预防组和EAMG组临床评分分别为(0·33±0·65)、(1·46±1·08)分,差异有统计学意义(P<0·01);预防组和EAMG组发病率分别为3/12(25%)和9/12(75%)只,差异有统计学意义(P<0·05)。(3)预防组和EAMG组TAChR特异性淋巴细胞增殖反应值分别为3347·6±556·9、9416·2±1546·5,预防组显著低于EAMG组(P<0·01)。(4)预防组和EAMG组淋巴细胞在TAChR刺激下分泌IFN-γ分别为(117·6±18·5)、(363·1±63·2)pg/ml,分泌IL-6为(49·7±9·9)、(90·6±13·6)pg/ml;与EAMG组相比,预防组淋巴细胞抗原特异性IFN-γ、IL-6分泌显著减少(P<0·01)。结论Tα146-162-iMDC能抑制TAChR预致敏T细胞免疫反应,预防EAMG的发生。Tα146-162-iMDC诱导免疫耐受与抗原特异性淋巴细胞增殖和IFN-γ、IL-6的分泌降低有关。
Objective To investigate the effect of Tα146-162-iMDC on immature myeloid dendritic cells (iMDC) carrying the immunodominant peptide α146-162 (Tα146-162) of acetylcholine receptor (TAChR) The preventive effect of EAMG and its mechanism. Methods (1) 24 C57BL / 6 mice were randomly divided into Tα146-162-iMDC group, iMDC group, control group and normal group, with 6 rats in each group. Tα146-162-iMDC group, iMDC group, and control mice were injected subcutaneously with TAChR antigen emulsion (on day 0). On day 7, Tα146-162-iMDC was injected subcutaneously in Tα146-162-iMDC group, and control iMDC and phosphate buffered saline (PBS) were injected in iMDC group and control group respectively. Normal mice do not interfere. On the 14th day, lymphocyte proliferation reaction was detected. (2) Another 24 C57BL / 6 mice were randomly divided into prophylaxis group and EAMG group. Each mouse was injected subcutaneously with antigen emulsion on days 0, 30 and 60 to induce EAMG. The Tα146-162-iMDC was injected subcutaneously in the prevention group three days after each immunization, and the control group injected with PBS subcutaneously. Observation of incidence and muscle strength score. The end of the first 90 days of observation. (3) 3H-TdR incorporation assay was used to detect the proliferation of lymphocytes. The levels of IFN-γ and IL-6 in the supernatant of lymphocyte culture medium were detected by enzyme-linked immunosorbent assay. Results (1) TAChR-specific lymphocyte proliferation response values (radioactive fluorescence scintillation counting) in Tα146-162-iMDC group, iMDC group, control group and normal group were 3314 · 1 ± 571 · 3 and 7717 · 5 ± 1283 · 4, respectively , 8608 · 6 ± 1458 · 9,922 · 9 ± 109 · 3, respectively. Compared with iMDC group and control group, Tα146-162-iMDC group had significantly lower antigen-specific lymphocyte proliferation reaction (P <0.01). (2) The clinical scores of the prevention group and the EAMG group were (0.33 ± 0.65) and (1.46 ± 1.08) points respectively, the difference was statistically significant (P <0.01); the prevention group and EAMG The incidence rates of the two groups were 3/12 (25%) and 9/12 (75%) respectively, the difference was statistically significant (P <0.05). (3) The TAChR-specific lymphocyte proliferation response values of the prevention group and the EAMG group were 3347.6 ± 556.9 and 9416.2 ± 1546.5, respectively, while the prevention group was significantly lower than that of the EAMG group (P <0.01). (4) The levels of IFN-γ secreted by T lymphocytes in the prophylaxis group and EAMG group were (117 · 6 ± 18 · 5) and (363 · ± 63 · 2) pg / ml and IL- · 7 ± 9 · 9 and (90 · 6 ± 13 · 6) pg / ml, respectively. Compared with EAMG group, the levels of IFN-γ and IL-6 in lymphocytes in prevention group were significantly decreased (P <0.01 ). Conclusion Tα146-162-iMDC can inhibit TAChR pre-sensitized T cell immune response and prevent EAMG. Tα146-162-iMDC induced immune tolerance and antigen-specific lymphocyte proliferation and IFN-γ, IL-6 secretion decreased.