多房棘球蚴感染对小鼠肝脾淋巴细胞及其亚群的影响

doi:10.12140/j.issn.1000-7423.2020.01.005

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (1): 30-35.doi: 10.12140/j.issn.1000-7423.2020.01.005

多房棘球蚴感染对小鼠肝脾淋巴细胞及其亚群的影响

章宁^1,2, 张传山², 李智德¹, 李亮¹, 王慧¹, 谭雅超¹, 尚琳琳¹, 温浩^1,2,*

1 新疆医科大学第一附属医院,中亚高发病病因预防治疗国家重点实验室,乌鲁木齐 830054;
2 新疆医科大学第一附属医院/临床医学研究院,新疆包虫病基础医学重点实验室,乌鲁木齐 830054

收稿日期:2019-07-08 出版日期:2020-02-28 发布日期:2020-03-19
通讯作者: 温浩, E-mail: dr.wenhao@163.com
作者简介:章宁(1990-),男,硕士研究生。从事肝脏损伤与修复的分子机制的研究。E-mail: xiaozhangdaifu@163.com
基金资助:
国家自然科学基金（No. 81560330,No. 81760368,No. 81660341）

The effects of Echinococcus multilocularis protoscoleces infection on lymphocytes and their subpopulations in mouse liver and spleen

ZHANG Ning^1,2, ZHANG Chuan-shan², LI Zhi-de¹, LI Liang¹, WANG Hui¹, TAN Ya-chao¹, SHANG Lin-lin¹, WEN Hao^1,2,*

1 State Key Laboratory of Pathogenesis, Prevention, Treatment of High Incidence Diseases in Central Asia, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China;
2 Xinjiang Key Laboratory of Echinococcosis, and WHO-Collaborating Center on Prevention and Care Management of Echinococcosis, Clinical Research Institute, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China

Received:2019-07-08 Online:2020-02-28 Published:2020-03-19
Contact: E-mail: dr.wenhao@163.com
Supported by:
Supported by the National Natural Science Foundation of China(No. 81560330, No. 81760368, No. 81660341)

摘要/Abstract

摘要： 目的探讨多房棘球蚴感染对C57BL/6小鼠肝和脾淋巴细胞及其亚群的影响。方法 32只C57BL/6小鼠随机分为4组,每组8只,对照组、低数量组（50个原头节/鼠)、中数量组(500个原头节/鼠)、高数量组(2 000个原头节/鼠),实验组取200 μl含不同数量多房棘球蚴原头节的混悬液,采用门静脉注射法建立多房棘球蚴感染小鼠模型,对照组注射等量生理盐水。感染后4周,每组各取4只小鼠,取肝脏组织,常规Masson染色,观察病理变化。剩余4只小鼠取肝脏和脾脏,制备肝、脾细胞悬液,流式细胞术检测肝、脾CD3⁺ T细胞、B细胞、NK细胞、NKT细胞、CD4⁺ T细胞、CD8⁺ T细胞、CD4⁺CD69⁺ T细胞、CD8⁺CD69⁺ T细胞绝对数以及初始型CD4⁺ T(CD4⁺ Tn)细胞、效应记忆性CD4⁺ T（CD4⁺ Tem）细胞、初始型CD8⁺ T（CD8⁺ Tn）细胞、效应记忆性CD8⁺ T（CD8⁺ Tem）细胞和中心记忆性CD8⁺ T（CD8⁺ Tcm）细胞比例的变化。检测数据采用Flow-Jo软件分析,并做流式图。采用Graphad Prism 7.0软件进行作图和统计学分析。结果感染后4周,对照组小鼠肝无明显变化,低、中数量组小鼠肝组织出现急性炎症反应减弱,部分病灶炎性反应逐渐转变为纤维化修复,形成小肉芽肿结节,呈点状或灶状坏死。高数量组小鼠肝内出现小囊泡,可见生发层结构,周围大量炎性细胞浸润。小鼠肝CD3⁺ T细胞绝对数高、中、低数量组和对照组分别为（4.10 ± 0.04）× 10⁵、（3.81 ± 0.14）× 10⁵、（3.02 ± 0.12）× 10⁵、（2.98 ± 0.03）× 10⁵,高数量组高于其他3组（P < 0.05或0.01）。小鼠脾CD3⁺ T细胞绝对数高、中、低数量组和对照组分别为（16.01 ± 0.40）× 10⁵、（11.03 ± 1.21）× 10⁵、（10.10 ± 1.01）× 10⁵、（9.71 ± 0.90）× 10⁵,高数量组高于其他3组（P < 0.01）。小鼠肝CD4⁺ T、CD8⁺ T细胞和脾B细胞、CD8⁺ T绝对数高数量组高于其他3组（P < 0.05或0.01）。小鼠肝CD4⁺CD69⁺ T细胞绝对数高、中、低数量组和对照组分别为（3.23 ± 0.10）× 10⁴、（1.98 ± 0.11）× 10⁴、（1.51 ± 0.26）× 10⁴、（1.19 ± 0.21）× 10⁴,高数量组高于其他组（P < 0.05）。小鼠脾CD4⁺CD69⁺ T细胞绝对数高、中、低数量组和对照组分别为（10.10 ± 0.41）× 10⁴、（8.91 ± 0.80）× 10⁴、（8.20 ± 0.41）× 10⁴、（6.81 ± 0.50）× 10⁴,高数量组高于对照组（P < 0.01）。肝、脾CD8⁺CD69⁺ T细胞绝对数高数量组高于其他3组（P < 0.05或0.01）。小鼠肝CD4⁺ Tn细胞比例高、中、低数量组和对照组分别为（15.52 ± 1.51）%、（19.3 ± 2.09）%、（20.66 ± 1.28）%、（23.62 ± 2.84）%,对照组高于其他组（P < 0.05或0.01）。肝、脾CD8⁺ Tn细胞比例对照组高于其他组（P < 0.05或0.01）。肝、脾CD8⁺ Tcm细胞比例高数量组高于对照组（P < 0.05）。结论高数量多房棘球蚴感染C57BL/6小鼠,肝区域以CD4⁺ T细胞和CD8⁺ T细胞募集为主,而脾以B细胞和CD8⁺ T细胞募集为主。

关键词: 多房棘球蚴, T淋巴细胞, 早期, 流式细胞术

Abstract: Objective To investigate the effect of Echinococcus multilocularis protoscoleces infection on lymphocytes and their subpopulations in livers and spleens of C57BL/6 mice. Methods Thirty-two C57BL/6 mice were randomly assigned into four groups of eight animals each: control group, low-dosage group (50 protoscoleces/mouse), mid-dosage group (500 protoscoleces/mouse) and high-dosage group (2 000 protoscoleces/mouse). The mice received portal vein injection of 200 μl protoscolex suspension or normal saline. Four weeks after infection, four mice in each group were euthanized to collect liver tissues for histopathological observation with routine Masson staining, while the other four mice were euthanized for orbital blood collection and liver and spleen cell suspension preparation. The numbers of CD3⁺ T cells, B cells, NK cells, NKT cells, CD4⁺ T cells, CD8⁺ T cells, CD4⁺CD69⁺ T cells, and CD8⁺CD69⁺ T cells, as well as the percentages of naive CD4⁺ T cells (CD4⁺ Tn), effector memory CD4⁺ T cells (CD4⁺ Tem), naive CD8⁺ T cells (CD8⁺ Tn), effector memory CD8⁺ T cells (CD8⁺ Tem) and central memory CD8⁺ T cells (CD8⁺ Tcm) were detected by flow cytometry (FCM). The FCM data were analyzed with Flow-Jo software. Statistical analysis was performed using Graphad Prism 7.0 software. Results Four weeks after infection, there were no significant changes in the livers of the control mice. Both the low- and mid-dosage groups showed weakening of acute inflammatory cell infiltration in liver tissues, and some lesions had gradually transformed to the fibrotic repair stage, with formation of small granulomatous nodules and appearance of penctate or focal necrosis. In the high-dosage group, small vesicles were seen in the liver. Germinal layers were observed, accompanied by intensive inflammatory cell infiltration. The numbers of CD3⁺ T cells in mouse liver in the high-, mid- and low-dosage groups and the control group were (4.10 ± 0.04) × 10⁵, (3.81 ± 0.14) × 10⁵, (3.02 ± 0.12) × 10⁵, and (2.98 ± 0.03) × 10⁵, respectively, with that in the high-dosage group being significantly higher than the other 3 groups (P < 0.05 or 0.01). The numbers of CD3⁺ T cells in mouse spleen in the high-, mid- and low-dosage groups and the control group were (16.01 ± 0.40) × 10⁵, (11.03 ± 1.21) × 10⁵, (10.10 ± 1.01) × 10⁵, and (9.71 ± 0.90) × 10⁵, respectively (P < 0.01), with that in the high-dosage group being significantly higher than the other 3 groups (P < 0.01). The high-dosage group also had significantly higher numbers of CD4⁺ T cells and CD8⁺ T cells in livers as well as B cells and CD8⁺ T cells in spleens, compared with other groups (P < 0.05 or P < 0.01). The numbers of CD4⁺CD69⁺ T cells in mouse liver in the high-, mid- and low-dosage groups and the control group were (3.23 ± 0.10) × 10⁴, (1.98 ± 0.11) × 10⁴, (1.51 ± 0.26) × 10⁴, and (1.19 ± 0.21) × 10⁴, respectively, with that in the high-dosage group being significantly higher than the other groups (P < 0.05). The respective numbers of CD4⁺CD69⁺ T cells in mouse spleen in the high-, mid- and low-dosage groups and the control group were (10.10 ± 0.41) × 10⁴, (8.91 ± 0.80) × 10⁴, (8.20 ± 0.41) × 10⁴, and (6.81 ± 0.50) × 10⁴, with that in the high-dosage group being significantly higher than the other groups (P < 0.01). The high-dosage group also had significantly higher numbers of CD8⁺CD69⁺ T cells in livers and spleens, compared with other groups (P < 0.05 or P < 0.01). The percentages of CD4⁺ Tn in mouse liver in the high-, mid- and low-dosage groups and the control group were (15.52 ± 1.51)%, (19.3 ± 2.09)%, (20.66 ± 1.28)%, and (23.62 ± 2.84)% respectively, with that in the control group being significantly higher than the other groups (P < 0.05 or P < 0.01). The control group also had significantly higher percentages of CD8⁺ Tn cells in livers and spleens, compared with other groups (P < 0.05 or P < 0.01). The high-dosage group had significantly higher percentages of CD8⁺ Tcm cells in livers and spleens, compared with other groups (P < 0.05). Conclusion In C57BL/6 mice infected with high-dose E. multilocular protoscoleces, the liver T cell recruitment occurred mainly of CD4⁺ and CD8⁺ T cells, while B cell and CD8⁺ T cell were mainly recruited in spleen.

Key words: Echinococcosis multilocularis, T cells, Early stage, Cytometry

中图分类号:

R532.21

章宁, 张传山, 李智德, 李亮, 王慧, 谭雅超, 尚琳琳, 温浩. 多房棘球蚴感染对小鼠肝脾淋巴细胞及其亚群的影响[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 30-35.

ZHANG Ning, ZHANG Chuan-shan, LI Zhi-de, LI Liang, WANG Hui, TAN Ya-chao, SHANG Lin-lin, WEN Hao. The effects of Echinococcus multilocularis protoscoleces infection on lymphocytes and their subpopulations in mouse liver and spleen[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(1): 30-35.

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多房棘球蚴感染对小鼠肝脾淋巴细胞及其亚群的影响

The effects of Echinococcus multilocularis protoscoleces infection on lymphocytes and their subpopulations in mouse liver and spleen

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