收稿日期: 2021-12-20
修回日期: 2022-03-20
网络出版日期: 2022-07-06
基金资助
上海市卫生和计划生育委员会青年基金(20214Y0206);上海市自然科学基金(19ZR1462600);国家自然科学基金面上项目(81971969);国家自然科学基金面上项目(81772225)
Preliminary study on the changes of plymorphonucler myeloid-derived suppressor cells in the spleen of mice infected with Schistosoma japonicum
Received date: 2021-12-20
Revised date: 2022-03-20
Online published: 2022-07-06
Supported by
Shanghai Health and Family Planning Commission Project for Youth(20214Y0206);Natural Science Foundation of Shanghai(19ZR1462600);the Surface Project of National Natural Science Foundation of China(81971969);the Surface Project of National Natural Science Foundation of China(81772225)
目的 初步探讨在日本血吸虫感染小鼠脾脏中,多核型髓源抑制细胞（PMN-MDSC）比例、功能及脾组织病理学的动态变化。 方法 36只BALB/c小鼠随机分为感染组和对照组,每组18只。感染组小鼠经皮感染日本血吸虫尾蚴（20 ± 1）条/鼠。感染后4、6、8周各组随机取6只小鼠取脾,称重并计算脾系数。固定、切片后,苏木精-伊红染色（HE）镜下观察脾组织病理变化。制备脾单细胞悬液,流式细胞术检测PMN-MDSC在脾淋巴细胞比例的动态变化。荧光定量PCR检测脾组织及脾PMN-MDSC相关炎症因子白细胞介素-6（IL-6）、S100钙结合蛋白A8（S100A8）、S100A9、细胞功能因子精氨酸酶1（Arg1）、一氧化氮合酶（iNOS）、血红素结合膜糖蛋白91（gp91）、转化生长因子-β（TGF-β）和IL-10的mRNA相对表达水平。采用SPSS 22.0统计学软件进行统计分析,组间比较采用独立样本t检验和ANOVA分析。 结果 感染后4、6、8周,小鼠脾质量分别为（179 ± 10.19）、（350.3 ± 16.84）、（414.3 ± 18.98）mg,脾系数分别为（0.93 ± 0.03）%、（1.97 ± 0.10）%、（2.31 ± 0.08）%,均高于对照组的（108.2 ± 9.93）mg、（0.51 ± 0.04）%（F = 101.3、143.7,P < 0.01）。切片脾组织HE染色镜下观察结果显示,和对照组相比,感染后4~6周,炎性细胞逐渐增加,脾淋巴滤泡减少,生发中心减少甚至消失;感染后6~8周,炎性细胞逐渐减少,脾脏淋巴滤泡、生发中心结构逐渐增生。感染后4、6、8周,脾PMN-MDSCs比例分别为（1.53 ± 0.16）%、（28.40 ± 2.35）%和（38.67 ± 1.94）%,高于对照组的（0.80 ± 0.10）%（F = 326.5,P < 0.01）。感染后6周,荧光定量PCR结果显示,脾组织中IL-6、S100A8、S100A9、gp91、Arg1、iNOS、IL-10和TGF-β基因的mRNA相对表达水平分别为2.74 ± 0.25、51.4 ± 1.25、39.20 ± 2.83、2.15 ± 0.08、2.33 ± 0.39、1.57 ± 0.08、2.20 ± 0.39和1.44 ± 0.05,均高于对照组的1.05 ± 0.10、1.01 ± 0.11、1.02 ± 0.07、1.04 ± 0.09、1.01 ± 0.06、1.00 ± 0.05、0.98 ± 0.20和1.00 ± 0.04（t = 6.367、40.07、13.50、9.311、3.315、5.642、2.764、6.914,P < 0.05或P < 0.01）;脾PMN-MDSC中iNOS因子的mRNA相对表达水平高于对照组 （t = 0.6.134,P < 0.01）。IL-10因子mRNA相对表达水平与对照组相比,差异无统计学意义（t = 1.176,P > 0.05）。感染后8周,脾组织中IL-6、S100A8、S100A9、Arg1、iNOS和IL-10的mRNA相对表达水平均高于对照组（t = 2.496、5.145、9.518、3.938、4.819、2.251,P < 0.05或P < 0.01）;脾PMN-MDSC中iNOS、IL-10因子mRNA相对表达水平分别为32.12 ± 2.30、2.64 ± 0.37,均显著高于感染后4周的1.08 ± 0.01、1.14 ± 0.35,感染后6周的12.06 ± 1.80、1.50 ± 0.36和对照组的1.02 ± 0.13、1.06 ± 0.12（F = 100.6、5.471,P < 0.01）。 结论 日本血吸虫感染后4~6周,脾组织炎症反应强烈;感染后6~8周,PMN-MDSC抑制因子IL-10分泌显著增高,脾组织结构也逐步恢复。
章孝成 , 高元 , 胡媛 , 曹建平 . 日本血吸虫感染小鼠脾多核型髓源抑制细胞变化的初步研究[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(3) : 330 -336 . DOI: 10.12140/j.issn.1000-7423.2022.03.008
Objective To explore the dynamic changes of the proportion, function and spleen histopathology of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the spleen of mice infected with Schistosoma japonicum. Methods Thirty-six BALB/c mice aged 6-8 weeks were randomly assigned into the infected group and control group, with 18 mice in each group. Mice in the infection group were infected with S. japonicum cercariae (20 ± 1)/mouse. At 4, 6 and 8 weeks post-infection, the spleen from 6 mice, which were randomly selected from each group, were collected, followed by calculating the spleen coefficients after weighting. The spleen tissues were fixed, sliced and stained with hematoxylin-eosin (HE) to observe the pathological changes microscopically. Spleen single cell suspension was prepared for examining the dynamic changes of PMN-MDSCs proportion in splenic lymphocytes by flow cytometry. Fluorescent quantitative PCR was used to determine the mRNA relative expression expression level of PMN-MDSC related inflammation factors, including interleukin-6 (IL-6), S100 calcium binding protein A8 (S100A8）, S100A9, and the cell function factor arginase 1 (Arg1), nitric oxide synthase (iNOS), heme-binding membrane glycoprotein 91(gp91), transforming growth factor-β(TGF-β), and IL-10 in the spleen tissues. Results At 4, 6 and 8 weeks after infection, the spleen weight and spleen coefficient in infection group were(179 ± 10.19)mg, (350.3 ± 16.84)mg, (414.3 ± 18.98)mg, and (0.93 ± 0.03)%, (1.97 ± 0.10)%, (2.31 ± 0.08)%, respectively, which were all significantly higher than that in the control group[(108.2 ± 9.93)mg and (0.51 ± 0.04)%] (F = 101.3, 143.7, P < 0.01). HE staining showed that at 4-6 weeks after infection, the inflammatory cell band gradually increased, while the lymphoid follicles of the spleen decreased, and the germinal center decreased or even disappeared compared with the control group. At 6-8 weeks after infection, the inflammatory cell band decreased gradually, and the lymphoid follicles and germinal centre structure of the spleen proliferated gradually compared with the control group. Splenic PMN-MDSCs proportion in infection group were (1.53 ± 0.16)%, (28.40 ± 2.35)%, (38.67 ± 1.94)%, which were all significantly higher than that in the control group (0.80 ± 0.10)% (F = 326.5, P < 0.01). Real-time quantitative PCR showed that at 6 weeks post-infection, the relative level of mRNA expression of IL-6, S100A8, S100A9, gp91, Arg1, iNOS, IL-10 and TGF-β in splenic tissue were 2.74 ± 0.25, 51.4 ± 1.25, 39.20 ± 2.83, 2.15 ± 0.08, 2.33 ± 0.39, 1.57 ± 0.08, 2.20 ± 0.39 and 1.44 ± 0.05, respectively, which were all significantly higher than that in the control group [1.05 ± 0.10, 1.01 ± 0.11, 1.02 ± 0.07, 1.04 ± 0.09, 1.01 ± 0.06, 1.00 ± 0.05, 0.98 ± 0.20 and 1.00 ± 0.04(t = 6.367, 40.07, 13.50, 9.311, 3.315, 5.642, 2.764, 6.914, P < 0.05, P < 0.01)]. The relative iNOS mRNA expression in splenic PMN-MDSCs was higher than that in the control group (t = 0.6134, P < 0.01) and the relative of IL-10 mRNA expression level was no statistically significantly different between the infection group and the control group(t = 1.176, P > 0.05). At 8 weeks after infection, the relative IL-6, S100A8, S100A9, Arg1, iNOS and IL-10 mRNA expression levels in splenic tissue were all significantly higher than those in the control group (t = 2.496, 5.145, 9.518, 3.938, 4.819, 2.251, P < 0.05, P < 0.01). In the splenic of PMN-MDSCs, the relative iNOS mRNA expression level and IL-10 were 32.12 ± 2.30, and 2.64 ± 0.37, respectively, which were higher than that at 4 weeks after infection (1.08 ± 0.01, 1.14 ± 0.35), and also higher than that at 6 weeks after infection (12.06 ± 1.80, 1.50 ± 0.36), as well as the control group (1.02 ± 0.13, 1.06 ± 0.12) (F = 100.6, 5.471, P < 0.01). Conclusion At 4~6 weeks post S. japonicum infection, the spleen tissue presented strong inflammatory response, at 6~8 weeks after infection, the secretion of the inhibitory factor IL-10 of PMN-MDSC was significantly increased, and the spleen tissue structure was gradually proliferated.
Key words： S. japonicum; PMN-MDSC; Spleen; Cytokine; Inflammation
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