肺结核患者外周血中Th细胞极化偏移及临床意义分析

目的:分析初诊肺结核患者外周血中CD4+T淋巴细胞及其亚型Th1和Th2细胞的改变,探讨其在肺结核病变过程中的临床意义。方法:取肝素抗凝全血,加RPMI1640培养液等体积混匀,依次加入1∶1 000稀释的PMA、1∶100稀释的Ion-omycin和1∶10稀释的Monensin,混匀后于37℃,5%CO2静置培养4小时或过夜。取100μl培养血细胞依次加入抗人CD3-Per-CP、CD8-APC、mIgG1-FITC、Rat IgG1-PE、IL-4-PE、IFN-γ-FITC抗体,按流式操作流程分别进行细胞膜和细胞浆内标记测定CD4+IL-4+(Th2)、CD4+IFN-γ+(Th1)两种细胞的水平。结果:初诊肺结核患者外周血中Th1水平均显著低于健康对照组(P0.01),而Th2水平则显著高于健康对照组(P0.05)。粟粒型肺结核患者外周血中Th1细胞含量显著低于浸润性肺结核患者(P0.05),而Th2水平显著高于浸润性肺结核和结核性胸膜炎(P0.05)。CD4+/CD3+T细胞比例在这三个病程中呈下降趋势,且粟粒型肺结核显著低于浸润性肺结核(P0.05)。糖尿病并肺结核患者Th1、CD4+/CD3+显著低于无糖尿病肺结核患者(P0.05),Th2含量则显著升高(P0.05)。15例重度肺结核患者经结核化疗与微卡治疗三个月后Th1、CD4+/CD3+水平较治疗前明显升高(P0.05),而Th2水平较治疗前显著降低(P0.01)。痰检或培养阳性与痰检阴性患者相比Th1、CD4+/CD3+水平呈下降趋势但无显著差异(P0.05),Th2细胞水平显著升高(P0.05)。结论:浸润性肺结核、结核性胸膜炎、粟粒型肺结核、糖尿病并肺结核患者存在着不同程度的免疫功能抑制,对Th1、Th2细胞水平与CD4+/CD3+比例测定有助于临床病情的判断和疗效观察。

Study on T helper cells in the patients of pulmonary tuberculosis and the clinical significance of Th polarization

Objective:This study was to analyze the changes of CD4~+ T lymphocytes and their subtypes,Th1 and Th2 cells,in the peripheral blood of patients with pulmonary tuberculosis disease and to investigate their clinical significance in the pathologic process of pulmonary tuberculosis.Methods:For polarization measurement of T-helper cells,1∶100 diluted Ionomycin and 1∶10 diluted Monensin were added in sequence into the equivalent volume mixture of heparin anti-coagulated whole blood and RPMI-1640 culture liquid.After being well mixed,the mixture was incubated at 5% CO_2,37℃ for 4 hours or overnight.To 100 μl of the mixture and in sequence,the antibodies of CD3-PerCP,CD8-APC,mIgG1-FITC,Rat IgG1-PE,IL-4-PE or IFN-γ-FITC were added.The stained CD4~+ IL-4~+ (Th2) and CD4~+ IFN-γ~+ (Th1) were analyzed by flow cytometry.Results:Compared with those from healthy controls,the peripheral blood of pulmonary tuberculosis patients contained significantly fewer Th1 (P＜0.01) but significantly more Th2 cells (P＜0.05).Th1 cells in the peripheral blood of the patients with miliary pulmonary tuberculosis were obviously fewer (P＜0.05) than in infiltrative pulmonary tuberculosis patients.The amount of Th2 cells in the peripheral blood of the patients with miliary pulmonary tuberculosis was significantly more (P＜0.05) than in either infiltrative pulmonary tuberculosis or tuberculous pleurisy patients.The ratio of CD4~+/CD3~+ tended to decrease in all these patients,and it was much lower (P＜0.05) in the patients of miliary pulmonary tuberculosis than in infiltrative pulmonary tuberculosis patients.Patients suffering from both pulmonary tuberculosis and diabetes had significantly lower levels of Th1 cells and CD4~+/CD3~+ (P＜0.05) and more Th2 cells,compared with those of pulmonary tuberculosis patients without diabetes.Levels of Th1 and Th2 cells restored significantly (P＜0.05) in 15 severe pulmonary tuberculosis patients after receiving tuberculosis chemotherapy and microcalorie theropy for three months.Patients with positive sputum examination tended to have decreased Th1 and CD4~+/CD3~+ (P＞0.05) and significantly increased Th2 (P＜0.05).Conclusion:Immunosuppression existed,in different extents,in patients of infiltrative pulmonary tuberculosis,tuberculous pleurisy,miliary pulmonary tuberculosis as well as patients with both pulmonary tuberculosis and diabetes.Analysis of Th1,Th2 and CD4~+/CD3~+ may be benefit for the judgments of disease conditions and therapeutic effects.