【作者】 谢莉；

【导师】 高微微； 郑素华； 马玙；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 2004， 硕士

【摘要】 目的 探讨三种结核分枝杆菌抗原刺激外周血单个核细胞(PBMC)后γ干扰素(IFN-γ)检测对于结核分枝杆菌潜伏感染及结核病的诊断意义，并与结核菌素皮试(TST)进行比较。对抗原刺激全血24小时后IFN-γ水平与抗原刺激PBMC 5天后IFN-γ水平进行比较。 方法 研究对象共分三组，活动性结核组(57例)，健康对照组(27例)，肿瘤组(29例)，分别用结核分枝杆菌纯蛋白衍生物(PPD)，早期分泌抗原靶6kDa蛋白(early secretary antigenic target 6kDa protein ESAT-6)，38kDa抗原与PBMC共同培养5天，然后采用酶联免疫吸附法(ELISA)检测培养上清液中IFN-γ的浓度，并分离细胞提取总RNA，采用逆转录聚合酶链反应(RT-PCR)测定IFN-γ mRNA的表达。部分标本同步进行全血+抗原和PBMC+抗原两种方法培养。 结果 在健康组中，TST与BCG接种史和排菌病人密切接触程度呈正相关关系(P值分别为0.047和0.041)，而以ESAT-6为抗原的IFN-γ水平在不同接触程度组中差异显著，在有无卡痕组差异不显著。抗原刺激后的IFN-γ浓度在结核组最高，肿瘤组最低。38kDa抗原刺激后的IFN-γ浓度对于结核病诊断的敏感性为64.9％，特异性为89.3％。仅有部分标本在RT-PCR后可见IFN-γ mRNA的表达，且其mRNA表达水平与上清液中IFN-γ浓度无显著相关关系。以PBMC为基础和以全血为基础的两种IFN-γ检测相关性良好(r=0.835，P(0.001)。 结论 ESAT-6刺激后的IFN-γ检测用于结核分枝杆菌潜伏感染的诊断可能优于TST。38kDa抗原刺激后的IFN-γ检测可用于结核病的辅助诊断。抗原刺激PBMC 5天后IFN-γ的转录和翻译水平不平行。全血+抗原培养方法有可能取代PBMC+抗原培养方法。更多还原

【Abstract】 Objective: To investigate the in-vitro interferon-gamma(IFN- Y )release assay based on different Mycobacterium tuberculosis(M.tb) antigens in the diagnosis of tuberculosis and of latent tubercular infections, and to compare it with the tuberculin skin test(TST). To compare the assay based on the peripheral blood mononuclear cells (PBMC) with that based on the whole blood. Methods: All subjects were divided into three groups: group 1, patients with tuberculosis (n=62); group 2, healthy (n=27); group 3, patients with cancer (n=29). The PBMC were co-cultured for 5 days with different antigens: purified protein derivitives (PPD) of tuberculin, early secretary antigenic target 6kDa protein (ESAT-6), 38kDa antigen. The protein levels of IFN- y were detected by ELISA. Total RNA was extracted from the PBMC after 5 days culture. The expression of the IFN-r mRNA was detected by RT-PCR. The two methods based on the PBMC or based on the whole blood were done at the same time in 10 subjects. Results: For healthy control, the TST is positive related to the history of BCG vaccination and the contact degree with sputum-positive TB patients (P=0.047, P=0.041 respectively). But the ESAT-6 based IFN-r release assay is only significantly related to the contact degree with sputum-positive TB patients, and there is no significant positive relation with the history of BCG vaccination. The in-vitro IFN- Y release level is highest in TB patients arid is lowest in cancer patients. The sensitivity and the specificity of the IFN- y release assay based on 38kDa are 64.9% and 89.3% for the diagnosis of tuberculosis. The expression of IFN-r mRNA was detected only in part of subjects using RT-PCR, and it did not correlated with the production of the IFN-r protein. The two methods based on PBMC and whole blood correlate well (r=0.835, P<0.001)Conclusions: The IFN-r release assay based on ESAT-6 may be better than TST when diagnosis of the latency of M.tb. The IFN-r release assay based on 38kDa may be a promising reagent when diagnosis to the tuberculosis. Expression of the mRNA for IFN-r does not parallel the production of the IFN-r protein. The assay based on the whole blood may be replace the assay based on PBMC.更多还原