【作者】 刘义；

【导师】 李琦； 张宗德；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 内科学， 2011， 博士

【摘要】 目的:检测初治肺结核患者血浆和痰液IFN-γ、IL-17、TNF-α、IL-8的含量及其mRNA的表达,观察初治肺结核患者血浆和痰液中上述细胞因子的变化及特点、分析相关影响因素、了解宿主抗结核分枝杆菌的免疫机制、探讨其临床应用价值,为揭示结核病的免疫学机制、辅助肺结核的诊断、评估抗结核治疗的疗效提供依据。方法:受试者为初治继发性肺结核患者44例,正常对照者43例,肺癌患者18例。用ELISA法分别检测三组研究对象的血浆和痰液IFN-γ、TNF-α、IL-8、IL-17的含量;用RT-PCR法分别检测三组研究对象PBMC和痰细胞中IFN-γ、TNF-α、IL-8、IL-17的mRNA表达;正常对照组内用酶联免疫斑点法试验(ELISPOT)判断结核分枝杆菌潜伏感染;用荧光定量PCR法检测肺结核患者痰液中的菌量负荷;对上述检测结果及临床相关因素进行统计分析,对四种细胞因子的诊断价值进行分析。结果:1.正常对照组血浆和痰液IFN-γ含量相接近(P>0.05),而血浆IL-17、TNF-α、IL-8含量均明显高于痰液,有统计学差异(P<0.01)。此外,其PBMC、痰细胞中IFN-γ、IL-17、IL-8的mRNA表达水平相接近(P>0.05),而PBMC中TNF-α的mRNA表达水平明显高于痰细胞,有统计学差异(P<0.05)。2.与正常对照相比,肺结核患者血浆IFN-γ、IL-8含量增高,IL-17、TNF-α的含量降低;痰液IFN-γ含量降低,IL-17、TNF-α、IL-8含量增高;此外,肺结核患者痰液中IL-17、TNF-α含量高于血浆,而IFN-γ、IL-8含量则低于血浆,均有统计学差异(P<0.05或0.01)。3.影响因素分析:(1)在正常对照组中,潜伏感染组和健康组血浆和痰液中IFN-γ、IL-17、TNF-α、IL-8四种细胞因子含量均相接近(P>0.05)。(2)肺结核患者PBMC中仅IFN-γ、IL-17的mRNA表达高于正常对照组(P<0.05),TNF-α、IL-8的mRNA表达与正常对照组相接近(P>0.05);肺结核患者痰细胞中四种细胞因子的mRNA表达与正常对照组相接近(P>0.05)。肺结核患者痰细胞和PBMC中四种细胞因子的mRNA表达相接近,无统计学差异(P>0.05)。(3)血浆IFN-γ(r=0.843)和IL-8(r=0.861)与菌量负荷呈正相关,血浆中IL-17(r=-0.816)、TNF-α(r=-0.788)与菌量负荷呈负相关(P<0.01);痰液IFN-γ(r=-0.893)与菌量负荷呈负相关(P<0.01),痰液IL-17、TNF-α、IL-8含量与菌量负荷呈正相关(r分别为0.756,0.631,0.889)(P<0.01)。(4)具有空洞病变的患者血浆和痰液四种细胞因子含量与无空洞的肺结核患者相接近(P >0.05)。(5)外周血淋巴细胞降低的患者痰液中IFN-γ、IL-8含量低于淋巴细胞正常的患者(P<0.01或0.05),而痰液中IL-17、TNF-α以及血浆中四种细胞因子则与淋巴细胞正常的患者相接近(P>0.05)。(6)抗结核治疗两周后,肺结核患者血浆IL-17、TNF-α含量增高,IL-8含量降低,有统计学意义(P<0.05);但血浆IFN-γ和痰液四种细胞因子含量无明显变化(P>0.05)。4.与肺癌患者相比,肺结核患者血浆IFN-γ、IL-8含量增高,IL-17、TNF-α的含量降低(P<0.01或P<0.05),痰液IFN-γ含量降低,IL-17、TNF-α含量增高(P<0.01或P<0.05);IL-8含量在两组间无统计学差异(P>0.05)。ROC曲线分析显示血浆和痰液IFN-γ、IL-17、TNF-α、IL-8含量的曲线下面积(AUC)在0.778～0.958之间(P<0.01),均为有效的诊断指标,其诊断的敏感度均大于68%,特异性均大于66%,诊断正确率均大于76%,其中,痰液TNF-α的敏感度最高,为90%;痰液IL-17的特异度最高,为97%;痰液IL-17的诊断正确率最高,为93%。各细胞因子血浆、痰液指标联合诊断可提高诊断的敏感度和特异性。结论:1.正常对照者代表全身免疫状态的血浆中IFN-γ和代表局部免疫状态的痰液中IFN-γ保持一致。然而,正常对照者血浆和痰液IL-17、TNF-α、IL-8含量的差异显示及其所参与的局部免疫水平低于全身免疫水平。2.IFN-γ、IL-17、TNF-α和IL-8与结核病的发生、发展密切相关。其中,肺结核患者血浆IFN-γ含量增高所介导的全身特异性免疫应答增强很难弥补其血浆TNF-α和IL-17降低所导致的相关特异性免疫应答降低,也很难拮抗IL-8增高所导致的免疫损伤。肺结核患者痰液IL-17、TNF-α和IL-8的增高、IFN-γ的降低可能既参与对巨噬细胞活化的抑制也参与免疫损伤的介导,与感染局部病变的形成密切相关。3.肺结核患者血浆和痰液IFN-γ、IL-17、TNF-α和IL-8的水平受多种因素的影响。其中,血浆中IFN-γ和IL-8 mRNA的高表达与其血浆含量增高相关;菌量负荷对血浆IFN-γ、IL-8的升高以及IL-17、TNF-α的降低有影响,菌量负荷对痰液IFN-γ的降低以及IL-17、IL-8、TNF-α的升高有影响;低水平的淋巴细胞对痰液中IL-8的降低和IFN-γ的增高有一定的影响;抗结核治疗可使患者血浆细胞因子水平趋于正常。而结核分枝杆菌潜伏感染对血浆和痰液中四种细胞因子无明显影响,在空洞的形成中也未见四种细胞因子的显著作用。4.肺结核患者血浆和痰液IFN-γ、IL-17、TNF-α和IL-8的检测具有一定的临床意义。其治疗前后血浆含量的改变提示动态监测或观察血浆中四种细胞因子的含量可早期评估抗结核治疗的疗效。应用ROC曲线所得到的四种细胞因子的界限值可作为肺结核、特别是菌阴肺结核的辅助诊断指标。更多还原

【Abstract】 Object: to assess the contents of interferon-gamma(IFN-γ), Tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8), interleukin-17 (IL-17) in the plasma and sputum of pulmonary tuberculosis patients with initial treatment, and the messenger RNA (mRNA) expression of these cytokines in patients with pulmonary tuberculosis(PTB); to observe the variations and features of these cytokines; to analyze the relevant influencing factors; to understand the immunologic mechanism of the anti-tuberculosis process; to estimate the value of the cytokines in clinical application; finally to provide the basis of the prehension of the immunological mechanisms of PTB, the auxiliary tuberculosis diagnosis, and the evaluation for the efficacy of anti-TB treatment.Method: The subjects were 44 patients with secondary pulmonary tuberculosis initially treated, and there were 43 normal controls and 18 lung cancer patients as control groups. To detect the levels of IFN-γ, IL-17, TNF-αand IL-8 in plasma and sputum of the three target groups with ELISA; to check out the expression of the mRNA in PBMC and cells in the sputum of the three groups by RT-PCR; in the normal control group, to distinguish the latent tuberculosis infection(LTBI) by Enzyme-Linked Immune spot (ELISPOT)assay; to check out the mycobacterial load in sputum of the patients with Fluorescent Quantitation Polymerase Chain Reaction(FQ-PCR); Statistical analysis was performed to analyze the results and the relevant factors, and the diagnostic value of the four cytokines were also analyzed.Result:1. In the normal control group, IFN-γlevels in Plasma and sputum was similar (P>0.05), while plasma IL-17, TNF-α, IL-8 levels were significantly higher than those in sputum (P<0.01). In addition, the expression of the mRNA in PBMC and in sputum cells of IFN-γ, IL-17, IL-8 were close (P>0.05), the mRNA expression of TNF-αin PBMC was significantly higher than that of sputum cells (P<0.05).2. Comparing with the normal control group, levels of IFN-γ、IL-8 in plasma of pulmonary tuberculosis patients were higher and levels of IL-17, TNF-αin plasma were lower; levels of IFN-γin sputum were lower and levels of IL-17、TNF-α、IL-8 in sputum of pulmonary tuberculosis patients were higher. In the pulmonary tuberculosis group, comparing with the levels in plasma, levels of IL-17 and TNF-αin sputum were higher and levels of IFN-γand IL-8 in sputum were lower(P<0.01 or P<0.05).3. Influencing factors: (1) In the normal control group, The contents of the four cytokines IFN-γ, IL-17, TNF-α, IL-8 in plasma and sputum from the LTBI group were closed to that of the healthy groups(P>0.05). (2)only the expression of the mRNA of IFN-γ, IL-17in PBMC from PTB patients were higher than those in the normal control group (P <0.05), the expression of the mRNA of TNF-α, IL-8 in PTB patients were similar to those in the normal control group (P> 0.05).(3) The contents of IFN-γ(r=0.843)and IL-8(r=0.861) in plasma had a positive correlation with the mycobacteria level, the plasma contents of TNF-α(r=-0.788) and IL-17(r=-0.816) had a negative correlation with the mycobacteria level (P<0.01); The contents of IFN-γ(r=-0.893) in sputum had a negative correlation with the mycobacteria level, the sputum TNF-α、IL-8、IL-17 contents had a positive correlation with the mycobacterial level, (r values was 0.631,0.889,0.756, respectively)(P<0.01).(4) The levels of the four cytokines in plasma and sputum from the PTB group with cavitation were close to those without it(P>0.05).(5) The contents of IFN-γand IL-8 in the sputum of those patients whose peripheral blood lymphocyte counts were lower, were lower than the group whose peripheral blood lymphocyte counts was normal(P<0.01 and P<0.05 respectively), while the contents of TNF-α、IL-17 in sputum carried no difference between the two groups(P>0.05).(6) For the PTB patients, two weeks after the treatment, the contents of the TNF-αand IL-17 in plasma were higher and the contents of the IL-8 in plasma were lower than before, which carried a statistical difference(P<0.05). The contents of the IFN-γin the plasma and the four cytokines in sputum were similar before and after the treatment(P>0.05).4. Comparing with the lung cancer group, the levels of IFN-γ, IL-8 in the plasma of PTB patients were higher, and those of IL-17 and TNF-αwere lower(P<0.01 or P<0.05); the level of IFN-αin their sputum was lower, while levels of IL-17 and TNF-αin their sputum were higher(P<0.01 or P<0.05); the contents of IL-8 in sputum carried no statistical difference between these two groups(P>0.05). Receiver operating characteristic curve (ROC curve) analysis suggested that when the contents of IFN-γ, TNF-α, IL-8 and IL-17 in plasma and sputum were transformed into the area under the curve (AUC) which were 0.778～0.958, it could be an operative diagnosis index(P<0.01). The sensitivities were all above 68%, specificities were all above 66% and accuracies were all above 76%. Among them, the sensitivity of sputum TNF-αwas the highest, 90%; IL-17 in sputum carried the highest specificity, 97%; IL-17 in sputum also carried the highest diagnostic accuracy, which was 93%. The combination indexes of the cytokines in plasma and sputum could improve the sensitivity and specificity of a diagnosis.Conclusion:1. In the normal control group, The level of IFN-γin plasma which represents the general immunity status is consistent with that in sputum that represents the local immunity status. However, differences between the levels of IL-17, TNF-α, IL-8 in plasma and in sputum suggested that their participation in the local immunity is lower than that in the systemic immunity.2. IFN-γ, IL-17,TNF-αand IL-8 were closely related to the incidence and the development of tuberculosis. Among them, the increase of specific immunity which resulted from the increasing level of IFN-γin the plasma of PTB patients can hardly compensate for the reducing level of some specific immunity resulting from the lessening level of TNF-αand IL-17. And it is also difficult to antagonize the immune damage caused by the increasing level of IL-8.The increasing levels of IL-17、TNF-αand IL-8 in the sputum of PTB patients, the reducing level of IFN-γare closely related to the local lesion formation in an infection site caused by Both inhibition of the activation of macrophages and mediation of the immune injury.3. The levels of IFN-γ、IL-17、TNF-αand IL-8 in plasma and in sputum of the patients with PTB were affected by many factors. Among them, IFN-γand IL-8’s high expressions of the mRNA in plasma was in correlation with the increasing level of plasma. Bacterial counts carried an influence on the increasing levels of IFN-γ、IL-8 in plasma and the reducing levels of IL-17、TNF-αin plasma, bacterial counts affected the reduction of IFN-γand the increase of IL-17,IL-8, TNF-αin sputum. Low levels of lymphocytes had a certain extent to the reducing level of IL-8 and the increase of IFN-γin sputum. Anti-tuberculosis treatment in patients can make the plasma cytokine levels to be nearly normal. The latent Mycobacterium tuberculosis infection had no significant effect on the four cytokines in plasma and sputum, and the prominent role of cytokines had not been seen on cavity formation.4. Detection of the levels of IFN-γ, IL-17, TNF-αand IL-8 in plasma and sputum in Patients with pulmonary tuberculosis carries some certain clinical significance. The changes of the cytokines levels in plasma before and after treatment indicate that dynamic monitoring or observation the four cytokines levels in plasma may serve as early assessment markers for the efficacy of anti-TB treatment. The cutoff values which we can get from the application of ROC curve can be used as diagnosis indicators for tuberculosis, especially for culture negative pulmonary tuberculosis.更多还原