1,25(OH)2D3诱导实验性自身免疫性重症肌无力大鼠免疫耐受的机制

doi:10.6040/j.issn.1671-7554.0.2015.266

山东大学学报（医学版） ›› 2015, Vol. 53 ›› Issue (8): 5-10.doi: 10.6040/j.issn.1671-7554.0.2015.266

1,25(OH)₂D₃诱导实验性自身免疫性重症肌无力大鼠免疫耐受的机制

李秀华¹, 李晓丽¹, 段瑞生¹, 朱梅佳¹, 曹莉莉², 李衍滨¹, 王思¹, 岳龙涛², 马庆海³, 刘菲¹

1. 山东大学附属千佛山医院神经内科, 山东济南 250014;
2. 山东大学附属千佛山医院医学研究中心, 山东济南 250014;
3. 山东大学附属千佛山医院检验科, 山东济南 250014

收稿日期:2015-03-11 出版日期:2015-08-10 发布日期:2015-08-10
通讯作者: 李秀华.E-mail:lxh731023@126.com E-mail:lxh731023@126.com
基金资助:
山东省优秀中青年科学家科研奖励基金(2008BS03012,2014BSB14078);山东省自然科学基金(ZR2010HM068)

Effects of 1,25(OH)₂D₃ on the induction of immune tolerance in Lewis rats with EAMG

LI Xiuhua¹, LI Xiaoli¹, DUAN Ruisheng¹, ZHU Meijia¹, CAO Lili², LI Yanbin¹, WANG Si¹, YUE Longtao², MA Qinghai³, LIU Fei¹

1. Department of Neurology, Qianfoshan Hospital Affiliated to Shandong University, Jinan 250014, Shandong, China;
2. Central Laboratory, Qianfoshan Hospital Affiliated to Shandong University, Jinan 250014, Shandong, China;
3. Department of Laboratory, Qianfoshan Hospital Affiliated to Shandong University, Jinan 250014, Shandong, China

Received:2015-03-11 Online:2015-08-10 Published:2015-08-10

摘要/Abstract

摘要： 目的探讨1,25-二羟维生素D₃[1,25(OH)₂D₃]对诱导实验性自身免疫性重症肌无力(EAMG)大鼠产生免疫耐受以及免疫耐受的机制.方法采用人工合成的大鼠来源乙酰胆碱受体α亚基97-116肽段(R97-116)免疫Lewis大鼠制备EAMG模型,随机分为1,25(OH)₂D₃治疗组(实验组)和对照组.进行临床症状评分、测量体质量,采用流式细胞术检测淋巴结MNC表型(CD80、CD86、MHC-II)和调节性T细胞的比例,采用CCK-8法测定特异性淋巴细胞增殖程度,采用流式细胞术测定细胞周期的分布,采用ELISA法检测淋巴结单核细胞(MNC)培养液中IL-4、IFN-γ、IL-17含量和血清中抗R97-116 IgG水平.结果从首次免疫后28 d开始,实验组临床症状评分显著低于对照组(P <0.01).从首次免疫后35 d开始,实验组体质量显著高于对照组(P <0.01).实验组淋巴结MNC表面CD80、CD86的表达较对照组显著降低(P <0.05).实验组淋巴结MNC中CD4⁺CD25⁺T细胞和CD4⁺FoxP3⁺ T细胞的表达显著高于对照组(P <0.01,P <0.05).实验组淋巴结MNC的淋巴细胞增殖反应较对照组显著降低(P <0.01).实验组淋巴结S期细胞的百分比显著低于对照组(P <0.05),S期和G2/M期细胞的百分比也显著低于对照组(P <0.05).实验组R97-116肽激活的淋巴细胞MNC上清液中IL-4水平较对照组显著增高(P <0.05),IFN-γ和IL-17水平较对照组显著降低(P <0.05).实验组血清中抗R97-116 IgG水平显著低于对照组(P <0.05).结论 1,25(OH)₂D₃通过诱导免疫耐受显著地改善EAMG大鼠的临床症状,其免疫耐受机制与降低MNC表面CD80和CD86的表达、上调调节性T细胞数量和Foxp3的表达、抑制淋巴细胞增殖、改变淋巴细胞周期、Th1/Th17型细胞反应向Th2型细胞反应的转化及降低抗R97-116 IgG抗体水平有关.

关键词: 1,25-二羟维生素D₃, 单核细胞, 细胞因子, 调节性T细胞, 抗R97-116 IgG, 实验性自身免疫性重症肌无力

Abstract: Objective To investigate the effects of 1,25(OH)₂D₃ on the induction of immune tolerance in Lewis rats with experimental autoimmune myasthenia gravis (EAMG). Methods EAMG models were established by subcutaneous injection of synthetic peptide R97-116 into both hind footpads of Lewis rats. The models were then divided into 1,25(OH)₂D₃ treatment group (experimental group) and control group. The clinical symptoms were evaluated. The expressions of CD80 and CD86 on MNC and Treg cells of lymph node mononuclear cells (MNC) were determined with flow cytometry. Cell proliferation was analyzed with CCK-8 test. Cell cycle was detected with flow cytometry. Cyto-kines of MNC supernatants were determined with ELISA. Serum anti-R97-116 IgG antibody was detected with ELISA. Results Compared with the control group, the experimental group exhibited lower clinical scores 28 d after first immunization (P <0.01) and less loss of body weight 35 d after first immunization (P <0.01); lower levels of CD80 and CD86 on MNC (P <0.05); higher percentages of CD4⁺CD25⁺ T cells and CD4⁺Foxp3⁺ T cells among lymph node MNC (P <0.01, P< 0.05); decreased lymphocyte proliferation in the presence of R97-116 antigen (P< 0.01); lower percentage of cells of S phase and of S phase plus G2/M phase (both P <0.05); higher level of IL-4 and lower levels of IFN-γ as well as IL-17 (all P <0.05); lower level of anti-rat R97-116 IgG antibody (P <0.05). Conclusion 1,25(OH)₂D₃ could ameliorate EAMG by down-regulating the expressions of CD80 and CD86 on MNC, up-regulating the number and function of Treg cells, inhibiting lymphocyte proliferation, changing the lymphocyte cell cycle, shifting cytokine profile from Th1/Th17 to Th2 type cytokines, and decreasing level of anti-R97-116 IgG antibody in serum.

Key words: Experimental autoimmune myasthenia gravis, Treg cells, Cytokines, 1,25(OH)₂D₃, Anti-R97-116 IgG, Mononuclear cells

中图分类号:

R746.103

李秀华, 李晓丽, 段瑞生, 朱梅佳, 曹莉莉, 李衍滨, 王思, 岳龙涛, 马庆海, 刘菲. 1,25(OH)₂D₃诱导实验性自身免疫性重症肌无力大鼠免疫耐受的机制[J]. 山东大学学报（医学版）, 2015, 53(8): 5-10.

LI Xiuhua, LI Xiaoli, DUAN Ruisheng, ZHU Meijia, CAO Lili, LI Yanbin, WANG Si, YUE Longtao, MA Qinghai, LIU Fei. Effects of 1,25(OH)₂D₃ on the induction of immune tolerance in Lewis rats with EAMG[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2015, 53(8): 5-10.

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1,25(OH)₂D₃诱导实验性自身免疫性重症肌无力大鼠免疫耐受的机制

Effects of 1,25(OH)₂D₃ on the induction of immune tolerance in Lewis rats with EAMG

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