中文摘要：

目的：探讨胁坍基因沉默的髓源树突状细胞（BMDC）负载电鳗乙酰胆碱受体（TAChR）优势肽段Tα146～162在TAChR预致敏C57BL／6小鼠中能否诱导免疫耐受。方法：制备产生RelBsiRNA分子的重组慢病毒和对照病毒。慢病毒感染BMDC后，给予LPS刺激，相应的DC命名为DC—siRelB和DC—control。应用实时定量PCR和Western印迹分析细胞中RelB表达，流式细胞仪检测细胞表型，ELISA检测上清白细胞介素（IL）-12水平。36只C57BL／6小鼠随机分为A1，A2，A3，K1，K2，K3共6组。实验前1天，A1-A3组用TAChR＋完全福氏佐剂（CFA）致敏；K1～K3组用钥孔戚血清蛋白（KLH）4-CFA致敏。第7天，A2和K2组注射Tα146-162脉冲的DC-siRelB；A3和K3组注射Tα146-162脉冲的DC—control；A1和K1组给予PBS。第14天，^3H-TdR掺入法检测淋巴细胞增殖反应。结果：成功构建了含RelB^shRNA基因的重组慢病毒，RelBsiRNA可显著下调DC中RelB的表达。与DC—control相比，DC—siRelB中CD80，CD86，MHCII表达及IL-12水平显著降低。与A1和A3组相比，A2组TAChR刺激下淋巴细胞增殖反应显著降低（P〈0．05），ConA和KLH刺激下淋巴细胞增殖反应差异无统计学意义（P〉0．05）。K1，K2和K3组在KLH刺激下淋巴细胞增殖反应差异均无统计学意义（P〉0．05）。结论：慢病毒介导的RelB基因沉默的BMDC可抵制成熟诱导反应并能在TAChR预致敏的C57BL／6小鼠中诱导抗原特异性免疫耐受，为研究其用于治疗重症肌无力奠定了基础。

英文摘要：

Objective To investigate whether RelB-silenced bone marrow-derived dendritic cells （BMDC） pulsed with torpedo acetylcholine receptor （TAChR） immuno-dominant peptide Tα146-162 can induce tolerance in T cells primed with TAChR. Methods Recombinant lentivirus that produced RelB siRNA and control lentivirus were prepared and used to infect BMDCs. The infected BMDCs were stimulated with LPS, and the resulting cells were designated as DC-siRelB or DC-control, respectively. The mRNA and protein expression of RelB were examined by quantitative real-time PCR and Western blot. Cell surface markers of DC were evaluated by flow cytometry. IL-12 in the supernatant was detected by ELISA. Mice were randomly divided into 6 groups： A1 , A2, A3, K1 , K2, and K3. On day 0, group A1 , A2, and A3 were primed with TAChR in CFA and group K1 , K2 and K3 were primed with KLH ＋ CFA. On day 7, group A2 and K2 were injected with Tα146-162 pulsed DC-siRelB, group A3 and K3 were injected with Tα146-162 pulsed DC-control, while A1 and K1 group received PBS at the same time. On day 14, lymphocyte proliferative response of the 4 groups were measured. Results Recombinant lentivirus including RelBshRNA genes was successfully constructed. RelB siRNA knocked down RelB expression in BMDCs obviously. Compared with DC-control, DC-siRelB expressed a significantly lower level of CDS0, CD86, and MHC class II on their surface, producing lower level of IL-12. Compared with group A1 and A3, lymphocyte proliferative response to TAChR of A2 group was suppressed significantly （ P 〈 0. 05 ） . No different lymphocyte proliferative responses to KLH and ConA were seen in group A1 , A2 and A3 （ P 〉 0. 05 ）. No different lymphocyte proliferative responses were seen in group K1 , K2 and K3 （ P 〉 0.05 ）. Conclu- sion Lentiviral-mediated RelB-silenced BMDCs are maturation resistant and can induce antigen-specific tolerance in TAChR primed C57BL/6 mice, which provides a basis for further study of their therapeutic potential in myasthenia