肺炎支原体感染评价方法的比较研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0464

生物技术通报 ›› 2025, Vol. 41 ›› Issue (1): 110-119.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0464

肺炎支原体感染评价方法的比较研究

李志强¹^,²(), 王吉英¹^,², 袁厅², 王佳², 韦艳娜², 王玉格¹^,², 李少丽⁴, 邵国青², 冯志新²^,⁵, 于岩飞²^,⁵()

1.湖南农业大学动物医学院，长沙 410125
2.江苏省农业科学院兽医研究所，南京 210014
3.怀化职业技术学院动物科技学院，怀化 418099
4.首都儿科研究所，北京 100020
5.南京农业大学动物医学院，南京 210095

收稿日期:2024-05-17 出版日期:2025-01-26 发布日期:2025-01-22
通讯作者: 于岩飞，女，博士，副研究员，研究方向：呼吸道病原菌的致病机制及防控技术；E-mail: yuyanfeihaha@163.com
作者简介:李志强，男，硕士研究生，研究方向：动物病原防控技术；E-mail: lizq1037@foxmail.com
基金资助:
湖南省教育厅科学研究项目(22B1055);湖南省自然科学基金项目(2022JJ50321);江苏省科协青年科技人才托举工程(JSTJ-2023-XH031);国家自然科学基金项目(32172860);国家自然科学基金项目(32102675)

Comparative Study on the Evaluation Methods for Mycoplasma pneumoniae Infection

LI Zhi-qiang¹^,²(), WANG Ji-ying¹^,², YUAN Ting², WANG Jia², WEI Yan-na², WANG Yu-ge¹^,², LI Shao-li⁴, SHAO Guo-qing², FENG Zhi-xin²^,⁵, YU Yan-fei²^,⁵()

1. Animal Medical College of Hunan Agricultural University, Changsha 410125
2. Veterinary Institute of Jiangsu Academy of Agricultural Sciences, Nanjing 210014
3. Animal Science and Technology College of Huaihua Vocational and Technical College, Huaihua 418099
4. Capital Institute of Pediatrics, Beijing 100020
5. Animal Medical College of Nanjing Agricultural University, Nanjing 210095

Received:2024-05-17 Published:2025-01-26 Online:2025-01-22

摘要/Abstract

摘要：

【目的】以小鼠为动物模型，分析肺炎支原体感染（Mycoplasma pneumoniae infection, MPI）的特性并对病变评价标准进行比较分析，为MPI统一评价方法的提出提供参考。【方法】利用BALB/c小鼠经滴鼻途径，两次感染5×10⁷ CCU的M. pneumoniae M129株，14 d后检测小鼠体重、肺脏大体病变、肺组织病理变化及其两种定量分析方法、肺组织病原载量、外周血T淋巴细胞亚群和血清中IL-6的表达水平等指标，分析M. pneumoniae感染特性并对病变评价标准进行比较分析。【结果】感染M129菌株14 d后，BALB/c小鼠血清中IL-6表达水平上升；肺脏出现明显的实变，肺组织切片可见大量中性粒细胞浸润，并可观察到肺泡间隔增厚。采用两种方法对肺脏病理切片的病变程度进行比较评分，发现美国胸科学会（American Thoracic Society, ATS）开发的肺损伤病理学评分系统相较于26分法能更为精细更加客观地反应肺部病变情况。感染导致小鼠炎症反应的发生并最终引起小鼠体重的显著降低。另一方面，肺部病原载量结果显示，M129株感染小鼠14 d后肺部病原载量极低；T细胞亚群流式检测分析显示，机体产生了偏向CD4⁺ T细胞的免疫，提示M. pneumoniae感染与机体免疫互相博弈，机体的免疫反应可能会逐渐清除肺部的病原。【结论】M. pneumoniae经鼻感染能导致BALB/c小鼠发生系统性炎症反应。对于肺组织病的评价，ATS肺损伤病理学评分系统相较于26分法更为精细客观，有利于不同研究之间的比较分析。

关键词: 肺炎支原体, 小鼠感染模型, 肺脏病变, 病理评分系统, 免疫清除

Abstract:

【Objective】In this study, mice were used as animal models to analyze the characteristics of Mycoplasma pneumoniae infection（MPI）and to compare and analyze the evaluation criteria of lesions, aiming to provide a reference for the development of a unified evaluation method for MPI.【Method】BALB/c mice were infected with the M. pneumoniae M129 strain at a concentration of 5×10⁷ CCU twice via nasal drip. After 14 d, the weight, gross lung lesions, pathological changes of lung tissue and two quantitative analysis methods, pathogenic load of lung tissue, T lymphocyte subsets in peripheral blood and the expressions of IL-6 in serum were detected. The infection characteristics of M. pneumoniae were analyzed and the evaluation criteria of lesions were compared and analyzed.【Result】After 14 d of infection with M129 strain, the level of IL-6 in BALB/c mice serum increased; significant consolidation occurred in the lungs, a large number of neutrophil infiltrates were observed in lung tissue sections, and alveolar septal thickening was observed. Two methods were used to compare the degree of lung lesions in lung pathological sections, and it was found that the pathological scoring system for lung injury proposed by the American Thoracic Society（ATS）reflected lung lesions more finely and objectively than the 26-score method. Infection led to the development of inflammatory responses in mice and eventually caused a significant reduction in mice body weight. On the other hand, the results of lung pathogen load showed that the lung pathogen load of mice infected with M129 strain was very low after 14 d. Flow cytometry analysis of T cell subsets showed that the body produced immunity biased towards CD4⁺ T cells, suggesting that M. pneumoniae infection and body immunity play a mutual game, and the body's immune response may gradually clear the lung pathogen.【Conclusion】M. pneumoniae causes systemic inflammation via nasal drip in BALB/c mice. ATS lung injury pathology scoring system is more precise and objective than the 26-score method for the evaluation of lung tissue lesions, which is conducive to the comparative analysis of different studies.

Key words: Mycoplasma pneumoniae, mouse infection model, lung lesions, pathology scoring system, immune clearance

李志强, 王吉英, 袁厅, 王佳, 韦艳娜, 王玉格, 李少丽, 邵国青, 冯志新, 于岩飞. 肺炎支原体感染评价方法的比较研究[J]. 生物技术通报, 2025, 41(1): 110-119.

LI Zhi-qiang, WANG Ji-ying, YUAN Ting, WANG Jia, WEI Yan-na, WANG Yu-ge, LI Shao-li, SHAO Guo-qing, FENG Zhi-xin, YU Yan-fei. Comparative Study on the Evaluation Methods for Mycoplasma pneumoniae Infection[J]. Biotechnology Bulletin, 2025, 41(1): 110-119.

图/表 14

参考文献 38

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指标 Parameter	每视野得分Score per field
指标 Parameter	0	1	2
A.肺泡腔内的中性粒细胞 Neutrophils in the alveolar space	无None	1-5	>5
B.间隙内的中性粒细胞 Neutrophils in the interstitial space	无None	1-5	>5
C.透明膜Hyaline membranes	无None	1	>1
D.充满空气空间的蛋白 Proteinaceous debris filling the airspaces	无None	1	>1
E.肺泡间隔增厚 Alveolar septal thickening	<2×	（2-4）×	>4×

指标 Parameter	每视野得分Score per field
指标 Parameter	0	1	2
A.肺泡腔内的中性粒细胞 Neutrophils in the alveolar space	无None	1-5	>5
B.间隙内的中性粒细胞 Neutrophils in the interstitial space	无None	1-5	>5
C.透明膜Hyaline membranes	无None	1	>1
D.充满空气空间的蛋白 Proteinaceous debris filling the airspaces	无None	1	>1
E.肺泡间隔增厚 Alveolar septal thickening	<2×	（2-4）×	>4×

评价指标Evaluating indicator	评价标准Evaluation criterion	得分Score
A.细支气管/支气管周围浸润 Peribronchiolar/bronchial infiltrates	无 None	0
	少量：<25% Few: <25%	1
	很多：25%-75% Many: 25%-75%	2
	大部分：>75% Majority: >75%	3
B.细支气管/支气管周围浸润的定性 Quality of peribronchiolar/ bronchial infiltrates	无：偶见极少量渗出物或大的支气管周围淋巴样肿物 None: Occasional minimal infiltrates or large peribronchial lymphoid	0
	轻度：异常，常有间断的环 Mild: Abnormal, often with interrupted collar	1
	中度：完整的环或新月形的环，小于5个细胞的厚度 Moderate: Complete collar or crescent collar with the thickness of < 5 cells	2
	重度：完整的环，大于5-10个细胞厚度 Severe: Complete collar with the thickness of > 5-10 cells	3
C. 细支气管/支气管腔渗出 Peribronchiolar/ bronchial luminal exudate	无 None	0
	轻度：≤25%腔闭合 Light: ≤25% lumen occlusion	1
	重度：>25%腔闭合Heavy: >25% lumen occluded	2
D.血管周围浸润 Perivascular infiltrate	无 None	0
	少量：<10% Few: <10%	1
	很多：10%-50% Many: 10%-50%	2
	大部分：>50% Majority: >50%	3
E.实质性肺炎 Parenchymal pneumonia	无 None	0
	轻度：斑块状实质性浸润 Light: Patchy parenchymal infiltrates	3
	重度：斑块状和融合的实质性浸润 Heavy: Patchy and confluent parenchymal infiltrates	5

评价指标Evaluating indicator	评价标准Evaluation criterion	得分Score
A.细支气管/支气管周围浸润 Peribronchiolar/bronchial infiltrates	无 None	0
	少量：<25% Few: <25%	1
	很多：25%-75% Many: 25%-75%	2
	大部分：>75% Majority: >75%	3
B.细支气管/支气管周围浸润的定性 Quality of peribronchiolar/ bronchial infiltrates	无：偶见极少量渗出物或大的支气管周围淋巴样肿物 None: Occasional minimal infiltrates or large peribronchial lymphoid	0
	轻度：异常，常有间断的环 Mild: Abnormal, often with interrupted collar	1
	中度：完整的环或新月形的环，小于5个细胞的厚度 Moderate: Complete collar or crescent collar with the thickness of < 5 cells	2
	重度：完整的环，大于5-10个细胞厚度 Severe: Complete collar with the thickness of > 5-10 cells	3
C. 细支气管/支气管腔渗出 Peribronchiolar/ bronchial luminal exudate	无 None	0
	轻度：≤25%腔闭合 Light: ≤25% lumen occlusion	1
	重度：>25%腔闭合Heavy: >25% lumen occluded	2
D.血管周围浸润 Perivascular infiltrate	无 None	0
	少量：<10% Few: <10%	1
	很多：10%-50% Many: 10%-50%	2
	大部分：>50% Majority: >50%	3
E.实质性肺炎 Parenchymal pneumonia	无 None	0
	轻度：斑块状实质性浸润 Light: Patchy parenchymal infiltrates	3
	重度：斑块状和融合的实质性浸润 Heavy: Patchy and confluent parenchymal infiltrates	5

组别 Group	编号 Numbering	D0体重 D0 Weight/g	D14体重 D14 Weight/g	体重变化 Weight change/g
M129	B59	19.0	15.4	-3.6
	B60	18.5	15.8	-2.7
	B70	18.0	14.7	-3.3
	B72	17.7	13.9	-3.8
	B73	19.2	16.5	-2.7
NC	B56	18.8	20.4	1.6
	B57	17.2	20.5	3.3
	B58	17.8	21.3	3.5
	B69	18.1	20.8	2.7
	B71	18.0	20.6	2.6

肺炎支原体感染评价方法的比较研究

Comparative Study on the Evaluation Methods for Mycoplasma pneumoniae Infection

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组别 Group	编号 Numbering	各指标得分Scores for each indicator					总分 Total score
组别 Group	编号 Numbering	A	B	C	D	E	总分 Total score
M129	B59	0	2	0	0	0.6	0.292
	B60	0	2	0	0	0.6	0.292
	B70	0	2	0	0	0.7	0.294
	B72	0	2	0	0	0.7	0.294
	B73	0	2	0	0	0.6	0.292
NC	B56	0	0.8	0	0	0	0.112
	B57	0	0.6	0	0	0	0.084
	B58	0	0.7	0	0	0	0.098
	B69	0	0.6	0	0	0	0.084
	B71	0	0.3	0	0	0	0.042

组别 Group	编号 Numbering	平均Ct值 Average Ct value	病原载量 Pathogen load/（CFU·mL^-1）
M129	B59	-	-
	B60	-	-
	B70	-	-
	B72	33.91	6.25
	B73
NC	B56	-	-
	B57	36.5	1.09
	B58	-	-
	B69	-	-
	B71	-	-

组别Group	编号Numbering	CD4⁺/CD3⁺	CD8⁺/CD3⁺	CD4⁺/CD8⁺
M129	B59	0.799	0.194	4.12
	B60	0.797	0.192	4.15
	B70	0.810	0.175	4.63
	B72	0.814	0.176	4.63
	B73	0.797	0.191	4.17
NC	B56	0.768	0.222	3.46
	B57	0.738	0.255	2.89
	B58	0.753	0.237	3.18
	B69	0.758	0.236	3.21
	B71	0.745	0.244	3.05

组别Group	编号Numbering	IL-6/（pg·mL^-1）
M129	B59	123.35
	B60	96.36
	B70	78.35
	B72	86.65
	B73	46.19
NC	B56	10.59
	B57	16.08
	B58	11.68
	B69	17.43
	B71	23.29

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