Sema4D在乙型肝炎肝硬化患者外周血T淋巴细胞和血清中的表达及意义

温雪; 何昱静; 袁倩倩; 李初谊; 卢利霞; 于晓辉; 张久聪

doi:10.3969/j.issn.1001-5256.2023.04.011

Sema4D在乙型肝炎肝硬化患者外周血T淋巴细胞和血清中的表达及意义

DOI: 10.3969/j.issn.1001-5256.2023.04.011

温雪^{1, 2},
何昱静¹,
袁倩倩¹,
李初谊¹,
卢利霞¹,
于晓辉^1, , ,,
张久聪^1, , ,

1.
中国人民解放军联勤保障部队第九四〇医院消化内科，兰州 730050
2.
宁夏医科大学临床医学院，银川 750000

基金项目:

国家自然科学基金(青年基金) (81500454);

甘肃省科技计划项目 (21JR7RA017);

甘肃省消化系统重症疾病临床医学研究中心 (20JR10RA107)

伦理学声明：本研究于2020年10月18日经由解放军联勤保障部队第九四〇医院伦理委员会审批，批号：2020KYL153，所有受试者均签署知情同意书。

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者以及与公开研究成果有关的利益冲突。

作者贡献声明：温雪负责实验设计、标本采集、数据分析及论文撰写；何昱静、袁倩倩、李初谊和卢利霞负责采集标本，分析数据；于晓辉、张久聪负责指导撰写论文并最后定稿。

详细信息

通信作者:
于晓辉, yuxiaohui528@126.com (ORCID: 0000-0002-8633-3281)

张久聪, zhangjiucong@163.com (ORCID: 0000-0003-4006-3033)

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出版历程
- 收稿日期: 2022-09-07
- 录用日期: 2022-10-09
- 出版日期: 2023-04-20

Expression of Sema4D in peripheral blood T cells and serum of patients with hepatitis B cirrhosis and its clinical significance

Xue WEN^{1, 2},
Yujing HE¹,
Qianqian YUAN¹,
Chuyi LI¹,
Lixia LU¹,
Xiaohui YU^{1
, ,
,},
Jiucong ZHANG^{1
, ,
,}

1.
Department of Gastroenterology, The 940^th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army, Lanzhou 730050, China
2.
School of Clinical Medicine, Ningxia Medical University, Yinchuan 750000, China

Research funding:

National Natural Science Foundation of China (Youth Foundation) (81500454);

Science and Technology Program of Gansu Province (21JR7RA017);

Gansu Provincial Clinical Research Center for Severe Disease of Digestive System (20JR10RA107)

More Information

Corresponding author: YU Xiaohui, yuxiaohui528@126.com (ORCID: 0000-0002-8633-3281); ZHANG Jiucong, zhangjiucong@163.com (ORCID: 0000-0003-4006-3033)

摘要

摘要: 目的研究乙型肝炎肝硬化患者外周血T淋巴细胞和血清中Sema4D的表达，分析其与临床指标的相关性。方法纳入2020年10月—2021年11月在联勤保障部队第九四〇医院就诊的20例慢性乙型肝炎(CHB)患者，68例乙型肝炎肝硬化患者以及20例健康对照者。根据Child-Pugh分级标准将乙型肝炎肝硬化患者分为Child-Pugh A级组(n=24)、B级组(n=24)和C级组(n=20)。采集患者外周血，分离血清及外周血单个核细胞(PBMC)，流式细胞术检测PBMC中膜结合型Sema4D(mSema4D)⁺CD4⁺T、mSema4D⁺CD8⁺T淋巴细胞的表达，酶联免疫吸附实验检测血清中可溶型Sema4D(sSema4D)的表达，分析其与病毒复制、肝脏炎症指标的相关性。符合正态分布的计量资料多组间比较用单因素方差分析，进一步两两比较采用LSD-t检验；非正态分布的计量资料多组间比较用Kruskal-Wallis H检验，进一步两两比较采用Mann-Whitney U检验；采用Spearman进行相关性分析。结果 mSema4D⁺CD4⁺T、mSema4D⁺CD8⁺T淋巴细胞的表达在CHB组、乙型肝炎肝硬化组和对照组3组间比较差异均有统计学意义(F值分别为43.092、13.344, P值均＜0.001)，进一步两两比较差异均有统计学意义(P值均＜0.05)。随着Child-Pugh分级的增加，mSema4D⁺CD4⁺T淋巴细胞、mSema4D⁺CD8⁺T淋巴细胞的表达水平逐渐降低(F值分别为14.093、17.154，P值均＜0.05)，进一步两两比较差异均有统计学意义(P值均＜0.05)。对照组、CHB组、乙型肝炎肝硬化组sSema4D含量分别为1.54(1.42~1.71)ng/mL、1.08(1.07~1.38)ng/mL、4.87(2.13~14.97)ng/mL，3组间比较差异有统计学意义(H=32.366，P＜0.001)，进一步两两比较差异均有统计学意义(P值均＜0.05)。Child-Pugh A级组、B级组、C级组sSema4D含量分别为2.42(0.59~5.65)ng/mL、4.92(2.75~12.73)ng/mL、14.18(4.59~18.43)ng/mL，3组间比较差异有统计学意义(H=11.889，P=0.003)，进一步两两比较差异均有统计学意义(P值均＜0.05)。sSema4D水平与乙型肝炎肝硬化患者的ALT、HBV DNA定量的表达水平均呈明显正相关(r值分别为0.294、0.430，P值均＜0.05)。结论 Sema4D在乙型肝炎肝硬化患者T淋巴细胞膜上低表达，在血清中高表达。sSema4D可能通过影响乙型肝炎肝硬化患者的ALT、HBV DNA水平，参与疾病的发生发展。
- 肝硬化 /
- 乙型肝炎 /
- Sema4D /
- T淋巴细胞
Abstract: Objective To investigate the expression of Sema4D in peripheral blood T cells and serum of patients with hepatitis B cirrhosis and its correlation with clinical indicators. Methods A total of 20 patients with chronic hepatitis B (CHB), 68 patients with hepatitis B cirrhosis, and 20 healthy controls who attended The 940^th Hospital of Joint Logistics Support Force of Chinese People's Liberation Army from October 2020 to November 2021 were enrolled. According to Child-Pugh class, the patients with hepatitis B cirrhosis were divided into Child-Pugh class A group with 24 patients, Child-Pugh class B group with 24 patients, and Child-Pugh class C group with 20 patients. After peripheral blood samples were collected to isolate serum and peripheral blood mononuclear cells (PBMCs), flow cytometry was used to measure the expression of membrane-bound Sema4D (mSema4D)⁺CD4⁺ T cells and mSema4D⁺CD8⁺ T cells in PBMCs, and ELISA was used to measure the expression of soluble Sema4D (sSema4D) in serum; their correlation with viral replication and liver inflammation markers was analyzed. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups, and the Mann-Whitney U test was used for further comparison between two groups; a Spearman correlation analysis was also performed. Results There were significant differences in the expression of mSema4D⁺CD4⁺ T cells and mSema4D⁺CD8⁺ T cells between the CHB group, the hepatitis B cirrhosis group, and the control group (F=43.092 and 13.344, both P < 0.001), while there were significant differences between any two groups (P < 0.05). The expression levels of mSema4D⁺CD4⁺ T cells and mSema4D⁺CD8⁺ T cells gradually decreased with increasing Child-Pugh class (F=14.093 and 17.154, both P < 0.05), and there were significant differences between any two groups (P < 0.05). The content of sSema4D was 1.54(1.42-1.71) ng/mL in the control group, 1.08(1.07-1.38) ng/mL in the CHB group, and 4.87(2.13-14.97) ng/mL in the hepatitis B cirrhosis group, with a significant difference between the three groups (H=32.366, P < 0.001) and between any two groups (P < 0.05). The content of sSema4D was 2.42(0.59-5.65) ng/mL in the Child-Pugh class A group, 4.92(2.75-12.73) ng/mL in the Child-Pugh class B group, and 14.18(4.59-18.43) ng/mL in the Child-Pugh class C group, with a significant difference between the three groups (H=11.889, P=0.003) and between any two groups (P < 0.05). In patients with hepatitis B cirrhosis, the level of sSema4D was positively correlated with the levels of alanine aminotransferase (ALT) and HBV DNA quantification (r=0.294 and 0.430, both P < 0.05). Conclusion Sema4D is lowly expressed on T cell membrane and highly expressed in serum of patients with hepatitis B cirrhosis, and sSema4D may be involved in the development and progression of hepatitis B cirrhosis by affecting the levels of ALT and HBV DNA.
- Liver Cirrhosis /
- Hepatitis B /
- Sema4D /
- T-Lymphocytes

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表 1 各组间CD3⁺CD4⁺T淋巴细胞、CD3⁺CD8⁺T淋巴细胞的百分比

Table 1. Percentages of CD3⁺CD4⁺T and CD3⁺CD8⁺T cells between groups

组别	例数	CD3⁺CD4⁺T淋巴细胞(%)	CD3⁺CD8⁺T淋巴细胞(%)
对照组	20	55.94±13.55	36.96±8.98
CHB组	20	37.67±11.48¹⁾	28.12±9.06¹⁾
乙型肝炎肝硬化组	68	29.20±8.62¹⁾²⁾	20.07±9.81¹⁾²⁾
F值		53.294	25.803
P值		＜0.001	＜0.001
注：与对照组比较，1)P＜0.05；与CHB组比较，2)P＜0.05。

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表 2 各组间mSema4D⁺CD4⁺T淋巴细胞、mSema4D⁺CD8⁺T淋巴细胞的百分比

Table 2. Percentages of mSema4D⁺CD4⁺T and mSema4D⁺CD8⁺T cells between groups

组别	例数	mSema4D⁺CD4⁺T淋巴细胞(%)	mSema4D⁺CD8⁺T淋巴细胞(%)
对照组	20	31.98±14.75	37.36±12.36
CHB组	20	47.26±14.12¹⁾	46.84±6.61¹⁾
乙型肝炎肝硬化组	68	17.18±12.43¹⁾²⁾	28.85±16.02¹⁾²⁾
F值		43.092	13.344
P值		＜0.001	＜0.001
注：与对照组比较，1)P＜0.05；与CHB组比较，2)P＜0.05。

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表 3 mSema4D在乙型肝炎肝硬化患者不同Child-Pugh分级中的表达

Table 3. Expression of mSema4D in different Child-Pugh classification groups of patients in the cirrhosis caused by hepatitis B

组别	例数	mSema4D⁺CD4⁺T淋巴细胞(%)	mSema4D⁺CD8⁺T淋巴细胞(%)
Child-Pugh A级组	24	25.10±11.86¹⁾²⁾	39.71±13.78¹⁾²⁾
Child-Pugh B级组	24	16.79±11.73²⁾	28.39±15.29²⁾
Child-Pugh C级组	20	8.16±6.59	16.39±8.91
F值		14.093	17.154
P值		＜0.001	＜0.001
注：与Child-Pugh B级组相比，1)P＜0.05；与Child-Pugh C级组相比，2)P＜0.05。

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Sema4D在乙型肝炎肝硬化患者外周血T淋巴细胞和血清中的表达及意义

DOI: 10.3969/j.issn.1001-5256.2023.04.011

通信作者:
于晓辉, yuxiaohui528@126.com (ORCID: 0000-0002-8633-3281)

张久聪, zhangjiucong@163.com (ORCID: 0000-0003-4006-3033)

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Expression of Sema4D in peripheral blood T cells and serum of patients with hepatitis B cirrhosis and its clinical significance

Corresponding author: YU Xiaohui, yuxiaohui528@126.com (ORCID: 0000-0002-8633-3281); ZHANG Jiucong, zhangjiucong@163.com (ORCID: 0000-0003-4006-3033)

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Sema4D在乙型肝炎肝硬化患者外周血T淋巴细胞和血清中的表达及意义

DOI: 10.3969/j.issn.1001-5256.2023.04.011

通信作者: 于晓辉, yuxiaohui528@126.com (ORCID: 0000-0002-8633-3281) 张久聪, zhangjiucong@163.com (ORCID: 0000-0003-4006-3033)

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Expression of Sema4D in peripheral blood T cells and serum of patients with hepatitis B cirrhosis and its clinical significance

Corresponding author: YU Xiaohui, yuxiaohui528@126.com (ORCID: 0000-0002-8633-3281); ZHANG Jiucong, zhangjiucong@163.com (ORCID: 0000-0003-4006-3033)

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通信作者:
于晓辉, yuxiaohui528@126.com (ORCID: 0000-0002-8633-3281)

张久聪, zhangjiucong@163.com (ORCID: 0000-0003-4006-3033)