多房棘球蚴分泌物抗原对脂多糖诱导小鼠骨髓来源的树突状细胞表型及功能的影响

李文登; 李超群; 胡旺; 徐凯; 庞明泉; 乜茹; 冯浩杰; 张占红; 刘处处; 樊海宁

doi:10.3969/j.issn.1001-5256.2022.03.021

多房棘球蚴分泌物抗原对脂多糖诱导小鼠骨髓来源的树突状细胞表型及功能的影响

DOI: 10.3969/j.issn.1001-5256.2022.03.021

青海大学附属医院肝胆胰外科，青海省包虫病重点实验室，西宁 810000

基金项目:

国家重点研发计划项目 (2017YFC0909900);

青海省科技厅项目 (2020-ZJ-Y01)

伦理学声明：本研究方案于2019年6月12日经由新疆医科大学第一附属医院伦理委员会审批通过，批号：20190531-03，符合实验室动物管理与使用准则。

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：李文登、樊海宁、胡旺负责课题设计，修改论文；李文登、庞明泉负责撰写论文；庞明泉、徐凯、李超群、刘处处、乜茹、冯浩杰、张占红参与收集、分析数据；樊海宁、徐凯、刘处处负责指导撰写文章并最后定稿。

详细信息

通信作者:
樊海宁，E-mail: fanhaining@medmail.com.cn

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出版历程
- 收稿日期: 2021-07-29
- 录用日期: 2021-09-23
- 出版日期: 2022-03-20

Effect of Echinococcus multilocularis secreted antigen on the phenotype and function of mouse bone marrow - derived dendritic cells induced by lipopolysaccharide

Qinghai Province Key Laboratory of Hydatid Disease Research, Department of Hepatopancreatobiliary Surgery, The Affiliated Hospital of Qinghai University, Xining 810000, China

Research funding:

National Key R&D Projects (2017YFC0909900);

Project of Qinghai Provincial Department of Science and Technology (2020-ZJ-Y01)

More Information

Corresponding author: FAN Haining, fanhaining@medmail.com.cn(ORCID: 0000-0001-5313-5732)

摘要

摘要: 目的探究不同浓度多房棘球蚴分泌物抗原(Em-sAg)对脂多糖(LPS)诱导小鼠骨髓来源的树突状细胞(BMDC)表型和功能的影响。方法小鼠骨髓腔中所分离的骨髓前体细胞，经小鼠重组粒细胞-巨噬细胞集落刺激因子(GM-CSF)刺激后形成BMDC，于正倒置显微镜下观察细胞形态。用流式细胞术鉴定BMDC纯度后，分为Control组、阳性对照组(LPS 1 μg/mL)、LPS+3 mg/mL Em-sAg组、LPS+1.5 mg/mL Em-sAg组、LPS+0.75 mg/mL Em-sAg组、LPS+0.375 mg/mL Em-sAg组共6组。流式细胞术检测各组BMDC表面分子(CD80、CD86、MHC-Ⅱ分子)的表达情况，ELISA法检测各组细胞因子IL-12p70的表达水平。满足正态分布的计量资料多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验表示。结果在正倒置显微镜下观察到，培养8~10 d的细胞可见毛刺样突起，细胞呈完全悬浮状态。流式细胞术检测发现，CD11c的阳性率均在70%以上，据此鉴定所培养的细胞大部分为BMDC。流式细胞术的进一步结果表明，与Control组相比，LPS组表面的细胞分子CD80、CD86、MHC-Ⅱ均显著增加(P值均 < 0.05)；与LPS组相比，LPS+3 mg/mL Em-sAg组、LPS+1.5 mg/mL Em-sAg组、LPS+0.75 mg/mL Em-sAg组、LPS+0.375 mg/mL Em-sAg组中BMDC的CD80显著减少(F=34.870，P < 0.001)，CD86、MHC-Ⅱ无明显减少(P值均＞0.05)。ELISA法的结果表明，不同组间的IL-12 p70的水平差异均有统计学意义(F=73.140，P < 0.05)，与Control组相比，LPS组刺激后IL-12p70的表达量显著升高，差异有统计学意义(P < 0.05)；与阳性对照组相比，LPS+3 mg/mL Em-sAg组、LPS+1.5 mg/mL Em-sAg组、LPS+0.75 mg/ml Em-sAg组、LPS+0.375 mg/mL Em-sAg组均降低IL-12p70的表达量(P值均 < 0.05)，且Em-sAg浓度越高，降低促炎症因子IL-12p70表达量越明显。结论不同浓度Em-sAg均可抑制LPS诱导的BMDC成熟度和促炎症细胞因子IL-12p70的表达。
- 棘球蚴病, 肝 /
- 树突细胞 /
- 脂多糖类 /
- 免疫耐受
Abstract: Objective To investigate the effect of different concentrations of Echinococcus multilocularis secretion antigen (Em-sAg) on the phenotype and function of mouse bone marrow-derived dendritic cells (BMDCs) induced by lipopolysaccharide (LPS). Methods The bone marrow precursor cells isolated from the mouse bone marrow cavity were stimulated by mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to form BMDCs, and then cell morphology was observed under an inverted microscope. After the purity of BMDCs was identified by flow cytometry, BMDCs were divided into control group, positive control group (LPS 1 μg/ml), LPS+3 mg/ml Em-sAg group, LPS+1.5 mg/ml Em-sAg group, LPS+0.75 mg/ml Em-sAg group, and LPS+0.375 mg/ml Em-sAg group. Flow cytometry was used to measure the expression of BMDC surface molecules (CD80, CD86, and MHC-Ⅱ molecules) in each group, and ELISA was used to measure the expression level of the cytokine IL-12p70. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results Observation under an inverted microscope showed that after 8-10 days of culture, the cells had burr-like protrusions and were in a state of complete suspension. Flow cytometry showed that the positive rate of CD11c was above 70% and most of the cultured cells were identified as BMDCs based on this. Flow cytometry further showed that compared with the control group, the LPS group had significant increases in the cell molecules CD80, CD86, and MHC-Ⅱ on surface (all P < 0.05); compared with the LPS group, the LPS+3 mg/ml Em-sAg group, the LPS+1.5 mg/ml Em-sAg group, the LPS+0.75 mg/ml Em-sAg group, and the LPS+0.375 mg/ml Em-sAg group had a significant reduction in CD80 (F=34.870, P < 0.001), while there were no significant reductions in CD86 and MHC-Ⅱ(P > 0.05). ELISA showed that there was a significant difference in the level of IL-12 p70 between groups (F=73.140, P < 0.05); compared with the control group, the LPS group had a significant increase in the expression level of IL-12p70 after stimulation (P < 0.05); compared with the positive control group, the LPS+3 mg/ml Em-sAg group, the LPS+1.5 mg/ml Em-sAg group, the LPS+0.75 mg/ml Em-sAg group, and the LPS+0.375 mg/ml Em-sAg group had a significant reduction in the expression level of IL-12p70 (P < 0.05), and the degree of reduction in the pro-inflammatory factor IL-12p70 increased with the increase in the concentration of Em-sAg. Conclusion Different concentrations of Em-sAg can inhibit LPS-induced maturity of BMDCs and the expression of the pro-inflammatory cytokine IL-12p70.
- Echinococcosis, Hepatic /
- Dendritic Cells /
- Lipopolysaccharides /
- Immune Tolerance

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图 1 原头节活性检测

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图 2 不同时期BMDC的形态变化

注：a，第0天(×20)；b，第3天(×20)；c，第6天(×20); d，第8天(×20)；e，第10天(×20)。

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图 3 流式细胞术鉴定BMDC的纯度

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图 4 流式细胞术检测不同浓度的Em-sAg干预LPS诱导的BMDC表面分子的表达情况

注：a，流式细胞术检测不同浓度的Em-sAg干预LPS诱导的BMDC的MHC-Ⅱ、CD80、CD86表达量；b，MHC-Ⅱ、CD80、CD86分子表达的定量分析。与Control组相比，*P < 0.05；与阳性对照组相比，**P < 0.05。

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图 5 不同浓度Em-sAg对LPS诱导的BMDC分泌IL-12p70的影响

注：与Control组相比，*P < 0.05；与阳性对照组相比，**P < 0.05。

下载: 全尺寸图片幻灯片

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