收稿日期: 2024-02-28
修回日期: 2024-03-26
网络出版日期: 2024-04-28
基金资助
国家自然科学基金(82372283);国家自然科学基金(82072307)
Study on the dynamic change of myeloid-derived suppressor cells proportion and cytokine expression in peripheral blood of mice infected with Echinococcus multilocularis
Received date: 2024-02-28
Revised date: 2024-03-26
Online published: 2024-04-28
Supported by
National Natural Science Foundation of China(82372283);National Natural Science Foundation of China(82072307)
目的 分析多房棘球蚴原头节感染小鼠外周血白细胞中髓源抑制性细胞（MDSC）及其亚型的比例动态变化，以及感染晚期小鼠血清中与MDSC增殖相关细胞因子的表达情况。方法 将24只BALB/c小鼠随机分为感染组和对照组，每组12只。感染组小鼠经腹腔注射1 200个原头节，对照组小鼠注射等量生理盐水。每组小鼠分别于感染后30、90和180 d随机各取3只，观察肝、脾组织形态学变化，并采集眼眶静脉丛外周血制备白细胞。外周血白细胞用外标抗体CD11b、Gr-1、Ly6G和Ly6C孵育后，流式细胞仪检测MDSC及其亚型多核MDSC（PMN-MDSC）和单核MDSC（M-MDSC）的占比。感染后180 d，采集感染组和对照组小鼠血清，ELISA检测白细胞介素6（IL-6）、IL-13、肿瘤坏死因子α（TNF-α）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）的浓度。使用GraphPad Prism 9.0软件进行制图与统计学分析，两组间比较采用独立样本t检验。结果 感染多房棘球蚴原头节后30 d，感染组小鼠肝组织出现囊泡，囊泡的体积随感染时间延长而增加；脾组织随感染时间延长逐渐肿大。流式细胞分析结果显示，感染后30、90和180 d，感染组小鼠外周血白细胞中MDSC占比分别为（13.2 ± 2.4）%、（15.7 ± 2.3）%和（41.3 ± 4.0）%，对照组小鼠的占比分别为（12.4 ± 3.2）%、（6.0 ± 0.9）%和（22.3 ± 1.1）%，感染后90和180 d感染组均高于对照组（t = 3.949、4.682，P < 0.05、0.01）；感染后30、90和180 d，感染组小鼠外周血白细胞中PMN-MDSC占比分别为（10.9 ± 2.1）%、（12.5 ± 2.4）%和（35.8 ± 3.6）%，对照组小鼠的占比分别为（9.6 ± 3.1）%、（4.5 ± 0.6）%和（18.5 ± 0.6）%，感染后90和180 d感染组均高于对照组（t = 3.237、4.788，P < 0.05、0.01）；感染后30、90和180 d，感染组小鼠外周血白细胞中M-MDSC占比分别为（1.8 ± 0.3）%、（1.1 ± 0.1）%和（4.6 ± 1.1）%，对照组小鼠的占比分别为（2.3 ± 0.2）%、（0.5 ± 0.1）%和（3.4 ± 0.9）%，感染后90 d感染组高于对照组（t = 3.246，P < 0.05）。感染后180 d，感染组小鼠血清中IL-6、IL-13、TNF-α和GM-CSF的浓度分别为（315.39 ± 13.58）、（339.41 ± 13.35）、（223.53 ± 27.49）和（262.31 ± 2.36） pg/ml，均高于对照组的（14.93 ± 0.55）、（50.74 ± 0.88）、（50.64 ± 1.64）和（115.28 ± 0.58） pg/ml（t = 22.100、21.580、6.277、60.460，P < 0.01或0.05）。结论 感染多房棘球蚴原头节后，小鼠外周血白细胞中MDSC比例随时间延长而增加，以PMN-MDSC增加为主。感染晚期小鼠血清中IL-6、IL-13、TNF-α和GM-CSF浓度升高，可能促进MDSC的增殖与活化。
苏雅馨 , 江楠 , 章孝成 , 王莹 , 蒋小凤 , 霍乐乐 , 王雅雪 , 曹建平 , 沈玉娟 . 多房棘球蚴感染小鼠外周血中髓源抑制性细胞比例动态变化及细胞因子表达研究[J]. 中国寄生虫学与寄生虫病杂志, 2024 , 42(2) : 211 -216 . DOI: 10.12140/j.issn.1000-7423.2024.02.012
Objective To analyze the dynamic changes in the proportion of myeloid-derived suppressor cells (MDSCs) and their subtypes in peripheral blood leukocytes of mice infected with Echinococcus multilocularis protoscoleces and detect the expression of cytokines related to MDSCs proliferation in the late-infected mice serum. Methods Twenty-four BALB/c mice were randomly divided into the infected group and the control group, with 12 mice in each group. Mice in the infected group were intraperitoneally injected with 1 200 E. multilocularis protoscoleces, while mice in the control group injected with the same volume of normal saline. Three mice from each group were randomly selected at 30, 90 and 180 days after infection to observe histomorphological changes in liver and spleen. Peripheral blood was collected from orbital venous plexus to prepare peripheral blood leukocytes. After incubation with externally labeled antibodies to CD11b, Gr-1, Ly6G and Ly6C, flow cytometry was used to detect the proportions of MDSCs, polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Serum samples from both groups were collected 180 days after infection for determine the concentrations of interleukin-6 (IL-6), IL-13, tumor necrosis factor α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) by ELISA assay. GraphPad Prism 9.0 software was utilized for graphical and statistical analysis; independent sample t-test was used for inter-group comparison. Results After E. multilocularis protoscoleces infection, cysts appeared in the liver of infected group mice 30 days after infection, and volume of cysts was increased over time. Spleen of infected group mice were enlaged over time 30, 90, and 180 days after infection. The results of flow cytometry showed that 30, 90 and 180 days after infection, the proportion of MDSCs in peripheral blood leukocytes of infected group were (13.2 ± 2.4)%, (15.7 ± 2.3)% and (41.3 ± 4.0)%, respectively. And the proportion of MDSCs in the control group were (12.4 ± 3.2)%, (6.0 ± 0.9)% and (22.3 ± 1.1)%, respectively. The infected group was significantly higher than the control group 90 and 180 days after infection (t = 3.949, 4.682; P < 0.05, 0.01). 30, 90 and 180 days after infection, the proportion of PMN-MDSCs in peripheral blood leukocytes of infected mice were (10.9 ± 2.1)%, (12.5 ± 2.4)% and (35.8 ± 3.6)%, respectively. And the proportion of PMN-MDSCs in the control group were (9.6 ± 3.1)%, (4.5 ± 0.6)% and (18.5 ± 0.6)%, respectively. The infected group was significantly higher than the control group 90 and 180 days after infection (t = 3.237, 4.788, P < 0.05, 0.01). 30, 90 and 180 days after infection, the proportion of M-MDSCs in peripheral blood leukocytes of infected mice were (1.8 ± 0.3)%, (1.1 ± 0.1)% and (4.6 ± 1.1)%, respectively. And the proportion of M-MDSCs in the control group were (2.3 ± 0.2)%, (0.5 ± 0.1)% and (3.4 ± 0.9)%, respectively. The infected group was significantly higher than the control group 90 days after infection (t = 3.246, P < 0.05). 180 days after infection, the concentrations of IL-6, IL-13, TNF-α and GM-CSF in the infected mice serum were (315.39 ± 13.58), (339.41 ± 13.35), (223.53 ± 27.49) and (262.31 ± 2.36) pg/ml, respectively, which were significantly higher than that of control group [(14.93 ± 0.55), (50.74 ± 0.88), (50.64 ± 1.64) and (115.28 ± 0.58) pg/ml] (t = 22.100, 21.580, 6.277, 60.460; P < 0.01, 0.01, 0.05, 0.01). Conclusion The proportion of MDSCs in peripheral blood leukocytes increased with time after E. multilocularis protoscoleces infection, mainly of PMN-MDSCs. The concentrations of serum IL-6, IL-13, TNF-α and GM-CSF increased of mice in the late stage of E. multilocularis protoscoleces infection, which may promote the proliferation and activation of MDSCs.
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