基于关键免疫细胞亚群的胆管癌患者外周血早期诊断标志物的研究

A Study of Early Diagnostic Markers in Peripheral Blood of Patients with Cholangiocarcinoma Based on Key Immune Cell Subsets

目的基于关键免疫细胞亚群探究胆管癌患者外周血早期诊断标志物。方法于2021年5月至2023年5月选取在本院确诊为CCA的患者75例即为CCA组,同期在本院收治的75例胆管炎患者即为对照组。同期在本院体检健康的志愿者75例为健康组。采用流式细胞仪检测CD3^(+)、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+)水平;采用酶联免疫吸附试验(ELISA)法检测肿瘤坏死因子(TNF-α)、白细胞介素-2(IL-2)、γ干扰素(IFN-γ)水平。采用Pearson法分析CCA患者CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)水平与TNF-α、IL-2、IFN-γ水平的相关性;ROC曲线分析外周血免疫细胞亚群对CCA发生的诊断价值。多因素Logistic回归分析CCA发生的影响因素。结果与健康组相比,对照组、CCA组患者CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)水平显著升高;与对照组相比,CCA组患者CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)水平显著升高(P<0.05)。与健康组相比,对照组、CCA组患者IL-2、IFN-γ水平显著降低,TNF-α水平显著升高;与对照组相比,CCA组患者IL-2、IFN-γ水平显著降低,TNF-α水平显著升高(P<0.05)。相关性分析显示,CCA患者CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)水平与IL-2、IFN-γ呈负相关关系,与TNF-α呈正相关关系(P<0.05)。外周血免疫细胞亚群(CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)、TNF-α、IL-2、IFN-γ)联合检测对CCA发生诊断的AUC显著高于单一指标诊断的AUC的值(Z_(CD3^(+)vs联合)=4.424,P<0.001;Z_(CD4^(+)vs联合)=3.425,P=0.001;Z_(CD4^(+)/CD8^(+)vs联合)=4.502,P<0.001;Z_(TNF-αvs联合)=3.322,P<0.001;Z_(IL-2^(+)vs联合)=7.473,P=0.001;Z_(IFN-γ^(+)vs联合)=3.166,P=0.001)。多因素Logistic回归分析显示,CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)、IL-2、IFN-γ水平是影响CCA发生的影响因素(P<0.05)。结论CCA患者外周血免疫细胞亚群水平显著升高,联合检测能够提高对CCA发生的诊断效能。

Objective To explore early diagnostic markers in peripheral blood of patients with cholangiocarcinoma based on key immune cell subsets.Methods From May,2021 to May,2023,75 patients diagnosed with CCA in our hospital were collected as the CCA group,while 75 patients with cholangitis admitted to our hospital were regarded as the control group.During the same period,75 healthy volunteers in our hospital were enrolled as the healthy group.Flow cytometry was applied to detect levels of CD3^(+),CD4^(+),CD8^(+),and CD4^(+)/CD8^(+);Enzyme linked immunosorbent assay(ELISA)was conducted to detect levels of tumor necrosis factor-α(TNF-α),interleukin-2(IL-2),andγ-interferon(IFN-γ).Pearson method was applied to evaluate correlations between CD3^(+),CD4^(+),CD4^(+)/CD8^(+)levels and TNF-α,IL-2,IFN-γlevels in CCA patients;ROC curve was drawn to evaluate values of peripheral blood immune cell subsets for the diagnosis of CCA.The influencing factors of CCA were identified by logistic regression.Results Compared with the healthy group,levels of CD3^(+),CD4^(+),CD4^(+)/CD8^(+)in control group and CCA group were significantly increased;Compared with the control group,levels of CD3^(+),CD4^(+)and CD4^(+)/CD8^(+)in CCA group were significantly increased(P<0.05).Compared with the healthy group,levels of IL-2 and IFN-γin the control group and CCA group were significantly decreased,while levels of TNF-αwere significantly increased;Compared with the control group,levels of IL-2 and IFN-γin CCA group were significantly decreased,while the levels of TNF-αwere significantly increased(P<0.05).Correlation analysis showed that the levels of CD3^(+),CD4^(+),CD4^(+)/CD8^(+)in CCA patients were negatively correlated with IL-2 and IFN-γ,but positively correlated with TNF-α(P<0.05).AUC value of the combined detection of peripheral blood immune cell subsets(CD3^(+),CD4^(+),CD4^(+)/CD8^(+),TNF-α,IL-2,IFN-γ)for the diagnosis of CCA was significantly higher than that of the single-marker diagnosis(Z_(CD3^(+)vs combination)=4.424,P<0.001;Z_(CD4^(+)vs combination)=3.425,P=0.001;Z_(CD4^(+)/CD8^(+)vs combination)=4.502,P<0.001;Z TNF-αvs combination=3.322,P<0.001;Z IL-2 vs combination=7.473,P=0.001;Z_(IFN-γvs combination)=3.166,P=0.001).Multivariate logistic regression analysis showed that levels of CD3^(+),CD4^(+),CD4^(+)/CD8^(+),IL-2,and IFN-γwere the influencing factors on the occurrence of CCA(P<0.05).Conclusion The level of peripheral blood immune cell subsets in patients with CCA is significantly increased,and the combination of these subsets could improve the diagnostic efficiency of CCA occurrence.