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中性粒细胞胞外诱捕网在原发性胆汁性胆管炎患者中的作用初步探析

偶绎颜 崔娜娜 李瑶 钱其炜 马雄 尤征瑞 连敏 王绮夏

引用本文:
Citation:

中性粒细胞胞外诱捕网在原发性胆汁性胆管炎患者中的作用初步探析

DOI: 10.3969/j.issn.1001-5256.2022.04.014
基金项目: 

国家自然科学基金项目 (82070581)

国家自然科学基金项目 (81800504)

国家自然科学基金项目 (81770564)

国家自然科学基金项目 (81790634)

上海市教育委员会和上海市教育发展基金会“晨光计划” (19CG16)

上海市卫生计生系统优秀青年医学人才培养计划 (2017YQ037)

上海市青年科技启明星计划 (18QA1402700)

伦理学声明:本研究取得所有受试者知情同意,并于2020年8月7日获得上海交通大学医学院附属仁济医院医学伦理委员会的批准,批号:SK2020-003。
利益冲突声明:本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明:偶绎颜负责所有实验和数据分析、撰写论文;崔娜娜负责收集分析病例资料和ELISA实验;李瑶、钱其炜参与数据收集;马雄、尤征瑞负责课题设计;王绮夏、连敏负责课题设计,指导撰写文章并最后定稿。
详细信息
    通信作者:

    连敏,sophialian24@163.com

    王绮夏,wqx0221155@126.com

    偶绎颜和崔娜娜对本文贡献等同,同为第一作者

A preliminary study of the role of neutrophil extracellular traps in patients with primary biliary cholangitis

Research funding: 

National Natural Science Foundation of China (82070581);

National Natural Science Foundation of China (81800504);

National Natural Science Foundation of China (81770564);

National Natural Science Foundation of China (81790634);

"Chen Guang" project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (19CG16);

the Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai (2017YQ037);

Shanghai Rising-Star Program (18QA1402700)

More Information
  • 摘要:   目的  探索原发性胆汁性胆管炎(PBC)患者外周血和肝组织内中性粒细胞胞外诱捕网(NET)表达水平,及其与临床生化指标的相关性。  方法  选取2016年8月—2020年8月上海交通大学医学院附属仁济医院收治的PBC患者24例、年龄匹配的原发性硬化性胆管炎(PSC)8例以及自身免疫性肝炎(AIH)患者19例、健康对照(HC)18例,分别检测血清中髓过氧化物酶(MPO)水平,并与临床指标进行相关性分析。利用组织免疫荧光检测PBC患者肝内NET表达情况。体外实验比较PBC患者和健康对照组外周血中性粒细胞产生NET能力的差异。满足正态分布的计量资料2组间比较采用独立样本t检验,非正态分布计量资料多组间比较采用Kruskal-Wallis H检验,2组间比较采用Mann-Whitney U检验。对MPO水平与肝脏相关实验室指标进行相关性分析,计算Spearman相关系数。  结果  PBC组血清中的MPO水平[811.21(450.67~1 216.20)ng/mL]较AIH组[468.58(142.63~812.43)ng/mL]和HC组[357.54(203.52~811.21)ng/mL] 明显升高(P值均<0.05);提示在PBC患者外周血中NET的产生量显著增加。PSC患者血清中MPO水平[763.56(489.59~1 633.14)ng/mL]也较HC组显著升高(P<0.05)。MPO水平与ALP(r=0.500,P<0.05)、GGT(r=0.426,P<0.05)、ALT(r=0.521,P<0.01)和AST(r=0.547,P<0.01)水平之间存在正相关关系。免疫荧光共聚焦显示PBC患者肝内存在H3Cit、MPO共定位。体外实验观察到PBC患者外周血中性粒细胞较健康对照在体外刺激时产生NET增多,且自发产生NET亦增高。  结论  PBC患者外周血及肝内NET增多,且外周血NET的含量与肝功能生化指标呈正相关。NET或可成为评估疾病严重程度的新型生物学标志物。

     

  • 图  1  外周血MPO水平与PBC相关自身抗体的相关性

    注:a,4组血清中MPO水平;b,PBC患者血清线粒体抗体(AMA)阳性或阴性者MPO水平;c,PBC患者血清抗2-丙酮酸脱氢酶(M2-3E)抗体阳性或阴性者MPO水平;d,PBC患者血清抗线粒体-M2型抗体(M2)阳性或阴性者MPO水平;e,PBC患者血清抗斑点蛋白抗体(sp100)阳性或阴性者MPO水平;f,PBC患者血清抗糖蛋白210抗体(gp210)阳性或阴性者MPO水平。

    Figure  1.  Correlation analysis between peripheral blood MPO levels and autoantibodies of PBC

    图  2  MPO水平与PBC患者肝功能和IgM的相关性分析

    Figure  2.  Correlation analysis of MPO level with liver function and immunoglobulin M in PBC patients

    图  3  肝组织中免疫荧光共聚焦定位NET

    注:a,PBC组;b,HC组;蓝色(DAPI);红色(MPO);绿色(H3Cit)。

    Figure  3.  Immunofluorescence confocal localization of NET in liver

    图  4  NET体外细胞荧光(×200)

    注:a,HC组外周血中性粒细胞空白组;b,PBC组外周血中性粒细胞空白组;c,HC组外周血中性粒细胞PMA组;d,PBC组外周血中性粒细胞PMA组。

    Figure  4.  In vitro cytofluorescence of NET(×200)

    图  5  HC组血清孵育后NET体外细胞荧光(×100)

    注:a,外周血中性粒细胞加10%健康人血清孵育后;b,外周血中性粒细胞加10%PBC患者血清孵育后。

    Figure  5.  In vitro cell fluorescence of NET after incubation with serum in HC group (×100)

    表  1  PBC患者肝脏生化指标基本资料

    Table  1.   Basic information of liver biochemical indicators in PBC patients

    指标 数值 与MPO相关性
    r P
    ALP (U/L) 124.50(83.00~194.50) 0.500 0.013
    GGT (U/L) 107.50(64.50~149.00) 0.426 0.038
    ALT (U/L) 31.00(22.10~61.00) 0.521 0.009
    AST (U/L) 36.00(21.65~52.50) 0.547 0.006
    TBil (μmol/L) 12.95(8.95~14.90) 0.380 0.067
    DBil (μmol/L) 4.40(3.15~6.35) 0.267 0.206
    IgM (g/L) 2.87(1.98~3.91) 0.007 0.976
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  • 收稿日期:  2021-11-30
  • 录用日期:  2022-01-08
  • 出版日期:  2022-04-20
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