活动性骨关节结核患者对潜伏感染相关蛋白Rv2659c T细胞免疫应答研究

T-Cell immunological properties of tuberculosis latent protein Rv2659c in active bone-joint tuberculosis

目的研究结核分枝杆菌（M.tb）潜伏感染相关蛋白Rv2659c对结核潜伏感染与活动性骨关节结核T细胞免疫应答的特性。方法使用CFP10-ESTA6蛋白（CE）通过酶联免疫斑点实验（ELISPOT）从健康志愿者中筛选出未感染健康组（42例）和结核潜伏感染组（33例）。从住院志愿者中筛选出非结核骨关节病组（35例）及活动性骨关节结核病组（30例）。使用Rv2659c蛋白刺激各组人群外周血单个核细胞（peripheral blood mononuclear cell,PBMC）,应用ELISPOT技术检测分泌IFN-γ的效应T细胞斑点数（斑点形成细胞,spot forming cells,SFC）,比较其在不同人群中的差异;以潜伏感染组为阳性组,以活动性骨关节结核病组为阴性组,绘制Rv2659c潜伏抗原的ROC曲线,在特异度80%及90%两个点取SFC的cut-off值,评价Rv2659c对结核潜伏感染与活动性骨关节结核的鉴别诊断效能。结果潜伏感染组的SFC值（2.5/28）显著高于活动性骨关节结核病组（0/2）、非结核骨关节病组（2.5/6.25）及未感染健康组（0/2.5）,均P〈0.05。Rv2659c重组蛋白的曲线下面积（AUC）为65.2%,95%可信区间（CI）0.51～0.794。特异度80%时,cut-off值为3;特异度90%时,cut-off值为5。Rv2659c蛋白诱导产生细胞免疫应答的阳性率在潜伏感染组最高（54.5%）,显著高于活动性骨关节结核病组（20%）、非结核骨关节病组（11.4%）以及未感染健康组（16.7%）3组,均P〈0.05。结论 Rv2659c诱导活动性骨关节结核患者效应T细胞产生的免疫应答显著低于结核潜伏感染人群;Rv2659c对于鉴别结核潜伏感染人群与活动性骨关节结核病具有一定诊断价值。

Objective To study the characteristics of T-cell immune response and differential diagnostic effectiveness of tuberculosis latent protein Rv2659 c in active bone-joint tuberculosis. Methods IFN-γ production from the peripheral blood mononuclear cell（ PBMC） was assayed by enzyme-linked immunospot assay（ ELISPOT） among healthy volunteers who consisted of uninfected healthy subjects（ 42 cases） and those with latent tuberculosis infection（ LTBI）（ 33 cases）.Non-tuberculosis bone disease patients（ 35 cases） and active bone-joint tuberculosis patients（ 30 cases） were filtered out among the inpatient volunteers. To define an ROC curve,the spot forming cell（ SFC） values stimulated by each antigen of LTBI subjects were taken as the positive group,and the active bone tuberculosis patients were used as a control group. The cut-off values were used to evaluate the performance of Rv2659 c in differential diagnosis of LTBI and active bone-joint tuberculosis. Results When stimulated by Rv2659 c,PBMC from LTBI subjects（ 2. 5 /28） yielded significantly higher ELISPOT counts than active bone-joint tuberculosis patients（ 0 /2）,non-tuberculosis bone disease patients（ 2. 5 /6. 25）and uninfected healthy subjects（ 0 /2. 5）（ P〈0. 05）. The area under the curve（ AUC） of Rv2659 c was up to 65. 2% and the 95 confidence interval（ CI） was 0. 51- 0. 794. When stimulated by Rv2659 c,the positive rate of latent infection（ 54. 5%） was higher than that among active bone-joint tuberculosis patients（ 20%）,non-tuberculosis bone disease patients（ 11. 4%） and uninfected healthy subjects（ 16. 7%）（ P〈0. 05）. Conclusion Rv2659 c is identified more easily by latent tuberculosis infected subjects and induces higher immune response via effector T cells compared with active bone-joint tuberculosis patients. Rv2659 c protein shows good performance in latent TB identification and active bone-joint tuberculosis differentiation.