细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞极化的研究

doi:10.12140/j.issn.1000-7423.2022.02.007

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (2): 175-180.doi: 10.12140/j.issn.1000-7423.2022.02.007

细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞极化的研究

孙叶挺(), 江楠, 姜岩岩, 李腾, 蒋小凤, 曹建平, 沈玉娟^*()

中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心）,国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,国家级热带病国际联合研究中心,上海 200025

收稿日期:2021-09-28 修回日期:2021-12-30 出版日期:2022-04-30 发布日期:2022-04-20
通讯作者: 沈玉娟
作者简介:孙叶挺(1997-),男,硕士研究生,从事棘球绦虫感染免疫研究。E-mail： 18863665089@163.com
基金资助:
国家自然科学基金(82072307);国家自然科学基金(81772224);国家科技重大专项(2018ZX10713001-004)

Study on the polarization of MDSC stimulated by Echinococcus granulosus protoscolex-derived exosomes in vitro

SUN Ye-ting(), JIANG Nan, JIANG Yan-yan, LI Teng, JIANG Xiao-feng, CAO Jian-ping, SHEN Yu-juan^*()

National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research);NHC Key Laboratory of Parasite and Vector Biology;WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai 200025, China

Received:2021-09-28 Revised:2021-12-30 Online:2022-04-30 Published:2022-04-20
Contact: SHEN Yu-juan
Supported by:
National Natural Science Foundation of China(82072307);National Natural Science Foundation of China(81772224);National Major Science and Technology Projects of China(2018ZX10713001-004)

摘要/Abstract

摘要：

目的探究细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞（MDSC）向M2型巨噬细胞极化的动态变化,并探讨二者协同发挥免疫抑制作用的生物学意义。方法体外培养细粒棘球蚴原头节并收集培养上清,超速离心上清获得外泌体。透射电镜观察外泌体形态,蛋白质免疫印迹（Western blotting）检测外泌体蛋白CD9和烯醇化酶的表达。取3只健康BALB/c小鼠,制备股骨髓系细胞,刺激分化为MDSC后,分为实验组、阳性对照组和阴性对照组。于6孔板中每孔加入1×10⁶个MDSC细胞,实验组、阳性对照组和阴性对照组分别加入20 μl外泌体（5 μg）、白细胞介素-4（IL-4）（40 ng）和RPMI 1640培养基进行刺激。于刺激24、48和72 h后,分别收集3组MDSC,采用流式细胞术分析单核型MDSC（M-MDSC）的细胞比例变化及其M2型巨噬细胞分子标志F4/80和CD206的表达情况。采用GraphPad Prism 8.0.2统计学软件进行统计学分析。结果透射电镜下可见细粒棘球蚴原头节源外泌体为圆形的膜状结构,直径集中在60~90 nm。Western blotting分析结果显示,外泌体有特异性蛋白CD9和烯醇化酶的表达。外泌体刺激MDSC 24 h后,实验组和阳性对照组M-MDSC占MDSC的比例分别为（77.14 ± 0.78）%、（75.74 ± 0.50）%,与阴性对照组的（78.13 ± 0.81）%比较差异均无统计学意义（t = 1.24、3.45,P > 0.05）;刺激48 h后,阴性对照组、实验组和阳性对照组M-MDSC占MDSC的比例分别为（80.47 ± 0.85）%、（78.27 ± 0.69）%和（68.01 ± 1.33）%,其中实验组与阴性对照组比较差异无统计学意义（t = 2.48,P > 0.05）,阳性对照组与阴性对照组比较差异有统计学差异意义（t = 11.20,P < 0.01）;刺激72 h后,实验组和阳性对照组M-MDSC占MDSC的比例分别为（50.03 ± 0.59）%和（46.14 ± 0.87）%,均低于阴性对照组的（74.94 ± 1.53）%,差异有统计学意义（t = 21.52、23.18,P < 0.01）。外泌体刺激MDSC 24 h、48 h和72 h后,实验组和阳性对照组M-MDSC上表达M2型巨噬细胞分子标志的比例分别为（11.83 ± 0.06）%、（12.48 ± 0.12）%,（15.11 ± 0.21）%、（15.42 ± 0.27）%和（29.12 ± 1.34）%、（31.83 ± 1.27）%,均高于阴性对照组的（8.52 ± 1.11）%、（8.79 ± 0.35）%和（15.49 ± 0.26）%,差异均有统计学意义（P < 0.05或P < 0.01）。结论细粒棘球蚴原头节源外泌体体外可调控M-MDSC向M2型巨噬细胞的极化,且极化随刺激时间进程不断推进。

关键词: 细粒棘球绦虫, 外泌体, 髓源抑制性细胞, M2型巨噬细胞

Abstract:

Objective To investigate the dynamic changes of the polarization of myeloid-derived suppressor cells (MDSC) to M2 macrophages stimulated by Echinococcus granulosus protoscolex-derived exosomes, and to explore the biological significance of their synergistic immunosuppressive effect. Methods The exosomes were collected by ultracentrifugation of supernatant of E. granulosus protoscoleces in vitro culture. The morphology of exosomes was observed by transmission electron microscope, and the expression of exosomal protein CD9 and enolase were detected by Western blotting. Subsequently, MDSC stimulated from myeloid cells of 3 healthy BALB/c mice femoral bone marrow were added at 1 × 10⁶ MDSC per well of a 6-well plate, and divided into experimental group, positive control group and negative control group. 20 μl exosomes (5 μg), 20 μl IL-4 (40 ng) and 20 μl RPMI 1640 medium were added respectively for co-culture. The proportions of monocytic DMSC (M-MDSC) and the expression of M2 macrophages molecular markers F480 and CD206 in co-cultured cells at 24 h, 48 h, and 72 h, were analyzed by Flow cytometry, respectively. Statistical analysis was performed by statistical software GraphPad Prism 8.0.2. Results E. granulosus protoscolex-derived exosomes are of circular membrane structure with a diameter of 60-90 nm by transmission electron microscopy. The expression of specific protein CD9 and enolase in exosomes was detected by Western blotting. The proportions of M-MDSC in MDSC in the negative control, experimental and positive control group were (78.13 ± 0.81)%, (77.14 ± 0.78)%, and (75.74 ± 0.50)%, respectively after 24 h of stimulation by exosomes. The differences were not significant (P > 0.05). The proportions of M-MDSC in the negative control, experimental and positive control group were (80.47 ± 0.85)%, (78.27 ± 0.69)%, and (68.01 ± 1.33)%, respectively, after 48 h of stimulation, showed no significant difference between experimental group and negative control group (P > 0.05), however significant difference was observed between the positive control and the negative control group (P < 0.01). After 72 h, the proportions of M-MDSC in the experimental and positive control groups in MDSC were (50.03 ± 0.59)% and (46.14 ± 0.87)%, which are lower than negative control group (74.94 ± 1.53)%, and the differences were statistically significant (P < 0.001). After stimulation with exosomes for 24 h, 48 h, and 72 h, the proportions of M-MDSC expressing M2 macrophages molecular markers in experimental and positive control group were (11.83 ± 0.06)%, (12.48 ± 0.12)%; (15.11 ± 0.21)%, (15.42 ± 0.27)%; and (29.12 ± 1.34)%, (31.83 ± 1.27)%, respectively, which were higher than those in the negative control group(8.52 ± 1.11)%, (8.79 ± 0.35)%, and (15.49 ± 0.26)%, and all differences were statistically significant (P < 0.05 or P < 0.01). Conclusion E. granulosus protoscolex-derived exosomes could regulate the polarization of M-MDSC to M2 macrophages, and the polarization continued to advance with the stimulation time.

Key words: Echinococcus granulosus, Exosomes, Myeloid-derived suppressor cells, M2 macrophages

中图分类号:

R383.33

孙叶挺, 江楠, 姜岩岩, 李腾, 蒋小凤, 曹建平, 沈玉娟. 细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞极化的研究[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 175-180.

SUN Ye-ting, JIANG Nan, JIANG Yan-yan, LI Teng, JIANG Xiao-feng, CAO Jian-ping, SHEN Yu-juan. Study on the polarization of MDSC stimulated by Echinococcus granulosus protoscolex-derived exosomes in vitro[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(2): 175-180.

图/表 4

参考文献 23

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细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞极化的研究

Study on the polarization of MDSC stimulated by Echinococcus granulosus protoscolex-derived exosomes in vitro

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