日本血吸虫病肝纤维化中肝星状细胞凋亡标志物的筛选

doi:10.12140/j.issn.1000-7423.2025.04.005

中国寄生虫学与寄生虫病杂志 ›› 2025, Vol. 43 ›› Issue (4): 475-481.doi: 10.12140/j.issn.1000-7423.2025.04.005

日本血吸虫病肝纤维化中肝星状细胞凋亡标志物的筛选

董博文(), 钟昊然, 朱丹霖, 李浩, 陆珂, 傅志强, 刘金明, 金亚美^*()

中国农业科学院上海兽医研究所，国家动物血吸虫病参考实验室，农业农村部动物寄生虫学重点实验室，上海 200241

收稿日期:2025-02-18 修回日期:2025-04-28 出版日期:2025-08-30 发布日期:2025-10-09
通讯作者: *金亚美，博士，研究员，从事寄生虫分子生物学与免疫学研究。E-mail：yameijin@shvri.ac.cn
作者简介:董博文，男，博士研究生，从事寄生虫与宿主相互作用研究。E-mail：dongbw131420@163.com
基金资助:
上海市“科技创新行动计划”启明星项目（扬帆专项）(24YF2756800);中国农业科学院科技创新工程(CAAS-ASTIP-2021-SHVRI);上海市自然科学基金(20ZR1469300)

Screening of marker of hepatic stellate cells apoptosis in schistosomiasis japonica-induced liver fibrosis

DONG Bowen(), ZHONG Haoran, ZHU Danlin, LI Hao, LU Ke, FU Zhiqiang, LIU Jinming, JIN Yamei^*()

National Reference Laboratory for Animal Schistosomiasis, Key Laboratory of Animal Parasitology of Ministry of Agriculture and Rural Affair, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China

Received:2025-02-18 Revised:2025-04-28 Online:2025-08-30 Published:2025-10-09
Contact: *E-mail: yameijin@shvri.ac.cn
Supported by:
Shanghai Sailing Program (Rising-Star Program) of the Science and Technology Innovation Action Plan(24YF2756800);Agricultural Science and Technology Innovation Program(CAAS-ASTIP-2021-SHVRI);Natural Science Foundation of Shanghai(20ZR1469300)

摘要/Abstract

摘要：

目的筛选适用于标记日本血吸虫病肝纤维化（SSLF）宿主肝星状细胞（HSC）凋亡的分子标志物。方法采用文献和数据库检索获取小鼠和日本血吸虫凋亡相关蛋白的氨基酸序列，利用NCBI网站的Protein BLAST功能对同源蛋白进行相似性分析。取感染日本血吸虫后第4、8、12周小鼠肝脏，以未感染小鼠为对照，制备肝脏切片，用免疫荧光法检测HSC标志物鼠抗肌间线蛋白（Desmin）和凋亡相关分子的表达情况，并分析感染后不同时间点小鼠肝脏HSC的凋亡情况，以筛选出适用于标记SSLF中宿主细胞凋亡的分子标志物。利用Jalview和MEGA11软件对日本血吸虫不同适应性宿主中该凋亡相关蛋白序列进行同源性和系统进化树分析。数据采用GraphPad Prism 8软件进行单因素方差。结果文献检索和数据库检索分别获得16个日本血吸虫凋亡相关蛋白和18个小鼠凋亡相关蛋白。序列相似性比对结果发现，小鼠与日本血吸虫的凋亡相关蛋白的序列相似性为20%~80%，日本血吸虫中未发现小鼠Fas和Fas配体（FasL）的同源蛋白。免疫荧光筛选结果显示，感染后8周，凋亡相关蛋白（绿色荧光）caspase-3剪切体（Cleaved caspase 3）、Bcl-2基因相关启动子（Bad）和FasL在虫卵肉芽肿周围皆有明显分布，其中Cleaved caspase 3、Bad除了可标记小鼠肝组织外，还与虫卵有强烈的特异性结合；FasL仅结合宿主的肝组织，与Desmin（红色荧光）有明显的共定位现象，且随着感染时间的延长，FasL与Desmin的共定位现象逐渐增多。FasL⁺Desmin⁺细胞（凋亡的HSC）占Desmin⁺细胞（HSC）的比例在感染后第4周为0，与未感染组（0）差异无统计学意义（P > 0.05）；感染后第8和第12周的占比分别为35.36%和89.12%，均高于未感染组（t = 13.38、28.77，P < 0.01）。不同宿主的FasL序列对比结果显示，小鼠FasL与大鼠、东方田鼠、人、猪、绵羊、水牛、马、犬、猫FasL的序列相似性分别为91.04%、80.57%、75.09%、76.24%、76.34%、77.99%、75.27%、74.48%和77.86%，具有同源性。系统进化树结果显示，与东方田鼠、人、猪、绵羊、水牛、马、犬、猫FasL相比，小鼠FasL与大鼠的亲缘关系较近。结论 FasL可用于标记日本血吸虫病肝纤维化宿主HSC的凋亡。

关键词: 日本血吸虫病, 肝纤维化, 肝星状细胞凋亡, Fas配体

Abstract:

Objective To screen molecular markers suitable for labeling host hepatic stellate cell (HSC) apoptosis in schistosomiasis japonica-induced liver fibrosis (SSLF). Methods Amino acid sequences of apoptosis-related proteins from Mus musculus and Schistosoma japonicum were obtained through literature review and database retrieval, and homology analysis of these proteins was performed using the Protein BLAST tool on the NCBI website. Liver tissues were collected from mice infected with S. japonicum 4, 8 and 12 weeks post-infection, and uninfected mice served as controls. Liver tissues were sectioned, and the expression of HSC marker Desmin and apoptosis-related molecules was detected using immunofluorescence staining. The dynamics of mouse HSC apoptosis were analyzed at different time points post-infection to identify molecular markers indicative of host cell apoptosis in SSLF. In addition, sequence homology and phylogenetic analysis of apoptosis-related proteins sequences from different adaptive hosts of S. japonicum were performed using the software Jalview and MEGA11. All data were analyzed with one-way analysis of variance (ANOVA) using the software GraphPad Prism 8. Results Sixteen S. japonicum apoptosis-related proteins and eighteen M. musculus apoptosis-related proteins were identified through literature review and database retrieval. Sequence similarity analysis showed 20% to 80% homology between sequences of M. musculus and S. japonicum apoptosis-related proteins, and no homologous proteins of M. musculus Fas or Fas ligand (FasL) were found in S. japonicum. Immunofluorescence staining showed remarkable distribution of apoptosis-related proteins (green fluorescence) Cleaved caspase 3, B cell-lymphoma-2 associated death promoter (Bad), and FasL surrounding egg granulomas in mice 8 weeks post-infection. Notably, Cleaved caspase 3 and Bad bound specifically to both mouse liver tissues and schistosome eggs, while FasL exclusively labeled host liver tissues, and was co-localized significantly with Desmin (red fluorescence). More importantly, the intensity of FasL and Desmin co-localization increased over time points post-infection. The proportion of FasL⁺Desmin⁺ cells (apoptotic HSCs) in Desmin⁺ cells (HSCs) was comparable between infected (0) and uninfected mice (0) 4 weeks post-infection (P > 0.05), and the proportions were 35.36% and 89.12% in infected mice 8 and 12 weeks post-infection, which were both significantly higher than in the uninfected group (t = 13.38, 28.77; both P < 0.01). Sequence alignment analysis showed that the homology of M. musculus FasL sequence was 91.04% with Rattus norvegicus, 80.57% with Microtus fortis, 75.09% with Homo sapiens, 76.24% with Sus scrofa, 76.34% with Ovis aries, 77.99% with Bubalus bubalis, 75.27% with Equus caballus, 74.48% with Canis lupus familiaris, and 77.86% with Felis catus. Phylogenetic analysis indicated that M. musculus FasL sequence was more closely related to R. norvegicus than to M. fortis, H. sapiens, S. scrofa, O. aries, B. bubalis, E. caballus, C. lupus familiaris, and F. catus. Conclusion FasL may be feasible tolabel host HSCs apoptosis in schistosomiasis japonica-induced liver fibrosis.

Key words: Schistosomiasis japonica, Liver fibrosis, Hepatic stellate cell apoptosis, Fas ligand

中图分类号:

R532.21

董博文, 钟昊然, 朱丹霖, 李浩, 陆珂, 傅志强, 刘金明, 金亚美. 日本血吸虫病肝纤维化中肝星状细胞凋亡标志物的筛选[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(4): 475-481.

DONG Bowen, ZHONG Haoran, ZHU Danlin, LI Hao, LU Ke, FU Zhiqiang, LIU Jinming, JIN Yamei. Screening of marker of hepatic stellate cells apoptosis in schistosomiasis japonica-induced liver fibrosis[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(4): 475-481.

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日本血吸虫凋亡蛋白 S. japonicum-derived apoptotic molecules	蛋白登录号 Accession no.	小鼠凋亡蛋白 M. musculus-derived apoptotic molecules	蛋白登录号 Accession no.	序列相似性/% Similarity/%
SjAIF	TNN08053.1	AIF	NP_001277293.1	32.75
SjAPAF	KAH8853392.1	APAF	NP_001269876.1	23.42
SjATM	KAH8866387.1	ATM	AAH67212.1	33.75
SjBad	AAW26442	Bad	NP_001272382.1	28.12
SjBak	CAX70134.1	Bak	NP_031549.2	36.00
SjBax	KAH8866109.1	Bax	NP_001398925.1	29.31
SjBcl2	KAH8856581.1	Bcl2	NP_033871.2	34.43
SjBIRP	TNN15698.1	BIRC6	NP_031592.3	60.19
SjCaspase 3	KAH8863222.1	Caspase 3	NP_001271338.1	39.38
SjCaspase 7	TNN10203.1	Caspase 7	NP_031637.1	40.40
SjCaspase 8	TNN08539.1	Caspase 8	NP_001073595.1	42.57
SjCaspase 9	Sjp_0071380	Caspase 9	NP_001264861.1	31.92
SjCIAP	TNN04927.1	Anamorsin	NP_598902.1	30.65
SjCyc	AAP06143.1	Cyc	AEP27250.1	80.00
/	/	Fas	NP_001140180.1	0
/	/	FasL	NP_034307.1	0
SjIAP	AAV97787.1	IAP1	NP_031490.2	46.00
Sjp53	TNN06778.1	TP53	NP_001120705.1	35.40
SjTNFR	TNN08705.1	TNF-R	NP_035739.2	30.14

日本血吸虫病肝纤维化中肝星状细胞凋亡标志物的筛选

Screening of marker of hepatic stellate cells apoptosis in schistosomiasis japonica-induced liver fibrosis

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