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慢性HBV感染者CD8+T细胞中Mg2+转运蛋白1 mRNA的表达及其与HBV DNA的相关性分析
DOI: 10.12449/JCH251111
伦理学声明：本研究于2024年5月27日由唐山市工人医院伦理委员会审批，批号：【2024】伦审研临第（048）号，患者均签署知情同意书。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：刘洋负责拟定写作思路，设计论文框架，撰写文章并最后定稿；林晓娥负责实验操作，研究过程的实施；孙如静、杨福玲负责样本数据收集，统计学分析和绘制图表；李树义负责制定研究方案，收集资料，数据分析与整理。
The mRNA expression of magnesium transporter 1 in CD8+ T cells and its correlation with HBV DNA in patients with chronic HBV infection
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摘要:
目的 探讨外周血CD8+T细胞中Mg2+转运蛋白1（MagT1）在慢性HBV感染者中的表达变化，并分析血清Mg2+、MagT1 mRNA与HBV DNA载量之间的关系。 方法 选取2022年1月—2023年12月唐山市工人医院收治的HBV感染者102例。采用病例对照设计，将受试对象依据疾病进展阶段划分为3组：慢性乙型肝炎患者40例、代偿期肝硬化患者32例、肝细胞癌患者30例；同时纳入年龄、性别匹配的健康志愿者32例作为正常对照。检测血清Mg2+浓度；使用实时荧光定量PCR检测CD8+T细胞中MagT1 mRNA表达水平及血清HBV DNA载量；采用流式细胞术定量分析CD8+T细胞表面程序性死亡受体1（PD-1）、T细胞免疫球蛋白黏蛋白-3（Tim-3）及自然杀伤细胞活化性受体2D（NKG2D）的表达水平。符合正态分布的计量资料多组间比较采用单因素方差分析，两两比较采用SNK-q检验；计数资料组间比较采用χ2检验；MagT1 mRNA与各指标的相关性采用Pearson线性相关分析。 结果 对照组、慢性乙型肝炎组、代偿期肝硬化组、肝细胞癌组血清Mg2+水平和MagT1 mRNA表达差异均有统计学意义（F值分别为29.014、145.578，P值均<0.001）。Pearson相关性分析显示，血清Mg2+浓度（r=0.335）和MagT 1 mRNA（r=0.394）表达与HBV DNA载量呈正相关（P值均<0.05）。对照组、慢性乙型肝炎组、代偿期肝硬化组、肝细胞癌组PD-1、Tim-3水平依次升高，NKG2D水平依次降低（P值均<0.001）。Pearson相关性分析显示，MagT1 mRNA表达与PD-1（r=-0.643）、Tim-3（r=-0.640）呈负相关，与NKG2D（r=0.655）呈正相关（P值均<0.05）。 结论 HBV感染者外周血CD8+T细胞MagT1 mRNA表达水平下降，且与血清HBV DNA载量呈正相关，MagT1表达降低可能通过阻碍Mg2+内流导致CD8+T细胞功能耗竭，表现为PD-1、Tim-3上调及NKG2D下调。 -
关键词:
- 乙型肝炎病毒 /
- CD8阳性T淋巴细胞 /
- 镁离子转运蛋白1
Abstract:Objective To investigate the change in the expression of magnesium transporter 1 （MagT1） in peripheral blood CD8+ T cells in patients with chronic hepatitis B virus （HBV） infection， as well as the correlation of serum Mg2+ and MagT1 with HBV DNA load. Methods A total of 102 patients with HBV infection who were admitted to Tangshan Workers’ Hospital from January 2022 to December 2023 were enrolled， and a case-control study was conducted. According to the stage of disease progression， the subjects were divided into chronic hepatitis B group with 40 patients， compensated liver cirrhosis group with 32 patients， and hepatocellular carcinoma group with 30 patients， and 32 healthy volunteers matched by age and sex were enrolled as normal control group. The serum concentration of Mg²⁺ was measured； RT-qPCR was used to measure the mRNA expression level of MagT1 in CD8+ T cells and serum HBV DNA load； flow cytometry was used to measure the expression levels of programmed death-1 （PD-1）， T-cell immunoglobulin and mucin-domain containing-3 （Tim-3）， and natural killer cell group 2D （NKG2D） on the surface of CD8+ T cells. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups， and the SNK-q test was used for comparison between two groups； the chi-square test was used for comparison of categorical data between groups； a Pearson linear correlation analysis was used to investigate the correlation between the mRNA expression level of MagT1 and other related indicators. Results There were significant differences in the serum level of Mg2+ and the mRNA expression level of MagT1 between the normal control group， the chronic hepatitis B group， the compensated liver cirrhosis group， and the hepatocellular carcinoma group （F=29.014 and 145.578， both P<0.001）. The Pearson correlation analysis showed that the serum level of Mg2+ and the mRNA expression level of MagT1 were positively correlated with HBV DNA load （r=0.335 and 0.394， both P<0.05）. The hepatocellular carcinoma group had the highest levels of PD-1 and Tim-3， followed by the compensated liver cirrhosis group， the chronic hepatitis B group， and the normal control group； the normal control group had the highest level of NKG2D， followed by the chronic hepatitis B group， the compensated liver cirrhosis group， and the hepatocellular carcinoma group （all P<0.001）. The Pearson correlation analysis showed that the mRNA expression level of MagT1 was negatively correlated with PD-1 and Tim-3 （r=-0.643 and -0.640， both P<0.05） and was positively correlated with NKG2D （r=0.655， P<0.05）. Conclusion There is a reduction in the mRNA expression level of MagT1 in peripheral blood CD8+ T cells in patients with HBV infection， which is positively correlated with serum HBV DNA load. The reduction in the expression of MagT1 may contribute to CD8+ T cell exhaustion by impairing Mg²⁺ influx， manifesting as the upregulation of PD-1 and Tim-3 and the downregulation of NKG2D. -
Key words:
- Hepatitis B Virus /
- CD8-Positive T-Lymphocytes /
- Magnesium Transporter 1
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图 1 HBV感染者Mg2+和MagT1 mRNA与HBV DNA的相关性
Figure 1. Correlation between Mg2+ and MagT1 mRNA and HBV DNA in HBV-infected individuals
图 2 HBV感染者MagT1 mRNA与CD8+T细胞表面分子的相关性
Figure 2. Correlation between MagT1 mRNA and the surface molecules of CD8+T cells in HBV-infected individuals
表 1 各组受试者一般资料比较
Table 1. Comparison of general information of subjects in each group
组别 例数 性别
（男/女，例）年龄
（岁）BMI
（kg/m2）对照组 32 18/14 40.50±8.10 23.22±2.10 慢性乙型肝炎组 40 20/20 38.52±7.52 22.45±2.55 代偿期肝硬化组 32 15/17 41.21±5.55 22.20±2.50 肝细胞癌组 30 15/15 43.20±8.33 21.89±2.55 统计值 χ2=0.801 F=2.328 F=1.704 P值 0.301 0.078 0.169 
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表 2 各组血清Mg2+浓度、MagT1 mRNA和HBV DNA水平比较
Table 2. Comparison of serum Mg2+ concentration， MagT1 mRNA and HBV DNA levels in each group
组别 例数 Mg2+（mmol/L） MagT1 mRNA HBV DNA（log10 IU/mL） 对照组 32 0.96±0.10 1.25±0.30 慢性乙型肝炎组 40 0.90±0.061） 0.80±0.251） 6.89±2.01 代偿期肝硬化组 32 0.86±0.061）2） 0.50±0.101）2） 5.33±1.502） 肝细胞癌组 30 0.80±0.051）2）3） 0.20±0.051）2）3） 4.33±1.202）3） F值 29.014 145.578 21.533 P值 <0.001 <0.001 <0.001 注：与对照组比较，1）P<0.05；与慢性乙型肝炎组比较，2）P<0.05；与代偿期肝硬化组比较，3）P<0.05。
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表 3 各组CD8+T细胞表面分子表达水平比较
Table 3. Comparison of surface molecular expression levels of CD8+T cells in each group
组别 例数 PD-1（%） Tim-3（%） NKG2D（%） 对照组 32 5.30±1.81 8.20±2.10 30.00±4.05 慢性乙型肝炎组 40 8.96±2.331） 12.34±3.401） 25.01±5.501） 代偿期肝硬化组 32 15.55±4.501）2） 20.32±5.501）2） 16.32±3.451）2） 肝细胞癌组 30 21.50±5.551）2）3） 28.34±5.851）2）3） 10.22±2.801）2）3） F值 120.815 130.010 140.699 P值 <0.001 <0.001 <0.001 注：与对照组比较，1）P<0.05；与慢性乙型肝炎组比较，2）P<0.05；与代偿期肝硬化组比较，3）P<0.05。
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