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代谢相关脂肪性肝病患儿外周血黏膜相关恒定T淋巴细胞的频率、表型及功能改变
DOI: 10.3969/j.issn.1001-5256.2023.03.012
伦理学声明：本研究方案于2020年4月30日经由湖南省儿童医院伦理委员会审批，批号：HCHLL-2020-38，所纳入患儿家属均签署知情同意书。
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：姜涛和谭艳芳负责课题设计，资料分析，撰写论文；康桢参与收集数据，修改论文；欧阳文献和李双杰负责拟定写作思路，指导撰写文章并最后定稿。
Changes in the frequency, phenotype, and function of mucosal-associated invariant T lymphocytes in peripheral blood of children with metabolic associated fatty liver disease
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摘要:
目的 探讨代谢相关脂肪性肝病(MAFLD)患儿外周血黏膜相关恒定T(MAIT)淋巴细胞的变化及其临床意义。 方法 选取2022年3月—2022年5月在湖南省儿童医院就诊的18例MAFLD患儿(MAFLD组)及同期20例正常对照儿童(对照组)，采集外周血，流式细胞仪检测MAIT淋巴细胞(CD3+CD161+TCRVα7.2+细胞)及其不同MAIT淋巴细胞亚型(CD4+CD8-MAIT淋巴细胞、CD4-CD8-MAIT淋巴细胞、CD4-CD8+MAIT淋巴细胞和CD4+CD8+MAIT淋巴细胞)比例，表达PD-1、CD69、穿孔素、CD107α、CXCR3、CXCR6和CCR6的MAIT淋巴细胞比例，分析MAIT淋巴细胞频率与肝脏炎症、脂肪含量和纤维化程度的相关性。符合正态分布的计量资料两组间比较采用t检验；非正态分布计量资料两组间比较采用Mann-Whitney U检验；相关性分析采用Spearman相关分析法。 结果 与对照组相比，MAFLD组中外周血MAIT淋巴细胞比例及表达PD-1、CD69、CD107α、CXCR3、CXCR6和CCR6的MAIT淋巴细胞比例均明显升高(P值均＜0.05)；MAIT淋巴细胞亚型中CD4+CD8-MAIT淋巴细胞、CD4+CD8+MAIT淋巴细胞所占MAIT淋巴细胞比例明显升高(P值均＜0.001)，CD4-CD8+MAIT淋巴细胞所占MAIT淋巴细胞比例降低(P＜0.001)；CD4+CD8+MAIT淋巴细胞(r=-0.474，P=0.047)和CD107α阳性MAIT淋巴细胞比例(r=-0.550，P=0.018)与ALT呈负相关。 结论 外周血MAIT淋巴细胞向肝脏趋化聚集，在儿童MAFLD肝脏炎症中发挥保护作用。 Abstract:Objective To investigate the change in mucosal-associated invariant T (MAIT) lymphocytes in peripheral blood of children with metabolic associated fatty liver disease (MAFLD) and its clinical significance. Methods A total of 18 children with MAFLD who attended Hunan Children's Hospital from March to May, 2022, were enrolled as MAFLD group, and 20 normal children who attended the hospital during the same period of time were enrolled as control group. Peripheral blood samples were collected, and flow cytometry was used to measure the percentages of MAIT lymphocytes (CD3+CDl61+TCRVα7.2+ cells), different MAIT lymphocyte subsets (CD4+CD8- MAIT cells, CD4-CD8- MAIT lymphocytes, CD4-CD8+ MAIT lymphocytes, and CD4+CD8+ MAIT lymphocytes), and MAIT lymphocytes expressing PD-1, CD69, perforin, CD107α, CXCR3, CXCR6, and CCR6. The correlation of MAIT lymphocyte frequency with liver inflammation, fat content, and fibrosis degree was analyzed. The t-test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. The Spearman correlation analysis was used for correlation analysis. Results Compared with the control group, the MAFLD group had significant increases in the percentage of MAIT lymphocytes in peripheral blood, the percentages of MAIT cells expressing PD-1, CD69, CD107α, CXCR3, CXCR6 and CCR6, and the percentages of CD4+CD8- MAIT cells and CD4+CD8+ MAIT lymphocytes among MAIT cell subsets (all P < 0.05), as well as a significant reduction in the percentage of CD4-CD8+ MAIT cells among MAIT cell subsets (P < 0.001). The percentages of CD4+CD8+ MAIT lymphocytes and CD107α-positive MAIT lymphocytes were negatively correlated with alanine aminotransferase (r=-0.474 and -0.550, P=0.047 and 0.018). Conclusion The migration of peripheral blood MAIT lymphocytes to the liver plays a protective role against liver inflammation in children with MAFLD. -
表 1 两组患儿临床资料比较
Table 1. The comparation of clinical data between the two groups
指标 MAFLD组(n=18) 对照组(n=20) 统计值 P值 男/女(例) 10/8 11/9 年龄(岁) 10.65(8.44~11.87) 9.00(7.40~11.25) Z=-1.083 0.279 ALT(U/L) 37.30(25.08~157.53) 13.65(11.75~17.00) Z=-4.474 ＜0.001 AST(U/L) 29.95(26.08~62.70) 20.95(18.90~24.23) Z=-3.304 0.001 WBC(×109/L) 7.31±2.56 7.00±1.50 t=0.937 0.355 中性粒细胞(×109/L) 4.47(3.36~5.18) 3.62(2.77~3.97) Z=-2.222 0.026 淋巴细胞(×109/L) 2.76(2.38~3.11) 2.44(1.82~2.91) Z=-1.608 0.112 
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表 2 两组患儿外周血MAIT淋巴细胞及各亚组MAIT淋巴细胞频率比较
Table 2. The comparation of the peripheral blood MAIT lymphocytes and subtypes between the two groups
指标 MAFLD组(n=18) 对照组(n=20) Z值 P值 MAIT淋巴细胞(%) 2.98(1.83~5.99) 0.29(0.09~0.69) -4.765 ＜0.001 CD4+CD8-MAIT淋巴细胞(%) 13.75(3.28~30.33) 0.38(0~1.55) -3.703 ＜0.001 CD4-CD8-MAIT淋巴细胞(%) 26.45(1.03~47.03) 31.25(24.05~50.33) 0.254 ＞0.05 CD4-CD8+MAIT淋巴细胞(%) 9.20(1.16~16.80) 43.65(29.65~64.98) -3.876 ＜0.001 CD4+CD8+MAIT淋巴细胞(%) 25.65(14.78~84.95) 9.36(6.04~22.45) -2.675 ＜0.001
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表 3 两组患儿外周血MAIT淋巴细胞表型和功能的差异
Table 3. Differences in phenotype and function of peripheral blood MAIT lymphocytes between the two groups
指标 MAFLD组(n=18) 对照组(n=20) Z值 P值 PD-1(%) 21.80(8.38~51.70) 4.43(2.07~11.29) -3.334 0.001 CD69(%) 56.00(28.93~68.35) 18.90(13.23~31.18) -2.646 0.008 穿孔素(%) 9.32(4.53~18.80) 4.16(1.35~9.53) -1.827 0.068 CD107α(%) 45.20(26.03~59.03) 10.95(6.80~17.18) -3.172 0.002 CXCR3(%) 87.35(80.08~96.03) 44.80(32.93~53.00) -4.678 ＜0.001 CXCR6(%) 30.40(8.24~54.90) 6.02(3.27~21.58) -2.939 0.003 CCR6(%) 26.80(13.03~77.70) 4.94(3.32~17.13) -2.749 0.006
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表 4 MAFLD组患儿外周血MAIT细胞频率与肝脏炎症、脂肪含量和纤维化程度相关性分析
Table 4. Correlation analysis between the frequency of MAIT lymphocytes in peripheral blood and the degree of liver inflammation, fat content, and fibrosis in children with MAFLD
项目 ALT AST 弹性值 脂肪肝参数 MAIT淋巴细胞 r值 0.041 -1.050 0.064 0.112 P值 0.871 0.677 0.801 0.660 CD4+CD8-MAIT淋巴细胞 r值 -0.305 -0.330 -0.303 -0.272 P值 0.218 0.181 0.221 0.275 CD4-CD8+MAIT淋巴细胞 r值 0.140 -0.130 -0.047 0.228 P值 0.580 0.606 0.854 0.363 CD4+CD8+MAIT淋巴细胞 r值 -0.474 -0.436 -0.347 -0.115 P值 0.047 0.071 0.158 0.650 CD107α阳性MAIT淋巴细胞 r值 -0.550 -0.334 -0.201 -0.168 P值 0.018 0.176 0.432 0.504
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