肺结核患者髓源性抑制细胞水平变化及其对T淋巴细胞增殖的影响研究

摘要

目的：探讨治疗前后肺结核（PTB）患者髓源性抑制细胞（MDSC）水平变化，分析 MDSC 对 T 淋巴细胞增殖的影响及其作用机制。方法选取2011年1月—2014年1月于临沂市人民医院诊断为活动性 PTB 患者30例，痰液涂片镜检结核分枝杆菌（MTB）阳性和结核菌素皮肤试验阳性（ TST +）的活动性 PTB 患者家属（接触 PTB 患者）12例，同期经本院结核内科检查为非结核病的健康对照者15例，同期本院痰液涂片镜检 MTB 阴性的肺部感染疾病患者7例。活动性 PTB 患者和接触 PTB 患者给予6个月的抗 PTB 治疗。取活动性 PTB 患者、接触 PTB 患者和健康对照者外周血制备单核细胞（ PBMC），取活动性 PTB 患者和肺部感染疾病患者肺部灌洗液制备支气管肺泡细胞（BALC），采用流式细胞仪进行流式分选，记录 MDSC 在 PBMC 和 BALC 中的比例。同样方法对治疗结束的活动性 PTB患者、治愈 PTB 患者和康复 PTB 患者检测 MDSC 在 PBMC 中的比例。从健康对照者分离的 PBMC 用 CFSE 活细胞荧光染色剂染色（CFSE - PBMC），从活动性 PTB 患者的 PBMC 中分离 CD +11b CD -14 MDSC，将不含 MDSC 的 PBMC 经丝裂霉素C 作用后培养。以2×105个 CFSE - PBMC 为反应细胞，2×105个不含 MDSC 的 PBMC 和 MDSC 以不同比例（1：0、1：2、1：4、1：8）混合作为刺激细胞共培养，测定 CD +3 T 淋巴细胞的 CFSE 信号强度。检测活动性 PTB 患者和健康对照者血清 L -精氨酸、一氧化氮（ NO）、鸟氨酸水平。结果活动性 PTB 患者、接触 PTB 患者、健康对照者 PBMC 中MDSC 比例分别为0.338（0.274，0.399）、0.115（0.073，0.201）和0.042（0.031，0.103），差异有统计学意义（H=16.450，P <0.001）；其中活动性 PTB 患者 PBMC 中 MDSC 比例高于接触 PTB 患者、健康对照者（P <0.001）。活动性 PTB 患者 BALC 中 MDSC 比例为0.425（0.219，0.483），高于肺部感染疾病患者的0.031（0.011，0.100）（ Z=9.675，P =0.004）。PBMC 与 MDSC 分别以1：0、1：2、1：4、1：8混合后，CD +3 T 淋巴细胞增殖率分别为0.874（0.761，0.953）、0.668（0.588，0.818）、0.510（0.477，0.614）、0.186（0.112，0.290），差异有统计学意义（H=9.995，P =0.006）。活动性 PTB 患者血清 L -精氨酸水平为31.72（18.37，59.58）μmol/ L，低于健康对照者的57.88（35.11，77.93）μmol/ L（Z =9.030，P =0.008）。活动性 PTB 患者血清 NO 水平为63.54（51.83，81.33）μmol/ L，高于健康对照者的30.31（28.19，40.28）μmol/ L（ Z =10.033，P =0.004）。治疗结束时治愈 PTB 患者PBMC 中 MDSC 比例为0.024（0.008，0.0389），低于活动性 PTB 患者治疗期间 PBMC 中 MDSC 的比例0.323（0.264，0.388）（Z =18.880，P <0.001）。康复 PTB 患者 PBMC 中 MDSC 比例为0.034（0.018，0.039），低于其治疗结束时PBMC 中 MDSC 的比例0.053（0.017，0.084）（Z =11.125，P =0.004）。结论活动性 PTB 患者 PBMC 和 BALC 中MDSC 比例增加，治愈及康复者 MDSC 比例下降；MDSC 可抑制 T 淋巴细胞增殖，其机制可能与激活 NO 信号通路有关。%Objective To investigate the level changes of myeloid - derived suppressor cell(MDSC)before and after treatment among patients with pulmonary tuberculosis( PTB),and to analyze the effects of MDSC on the proliferation of T lymphocytes and it′ s mechanism. Methods 30 active PTB patients,and 12 sputum smear microscopy MTB positive and tuberculin skin test positive(TST + )family members of active PTB patients(contacted PTB patients)who were diagnosed in Linyi People′s Hospital during January 2011 to January 2014,15 healthy controls who were excluded PTB patients in Department of Internal Medicine Tuberculosis of Linyi People′ s Hospital,and 7 sputum smear microscopy MTB negative patients with pulmonary infectious diseases during the same period,were selected as study subjects. The active PTB patients and contacted PTB patients were given anti - PTB therapy for 6 months. Peripheral blood from patients with active PTB,contacted PTB and healthy control was used to prepare peripheral blood mononuclear cell(PBMC),and lung lavage fluid from patients with active PTB and pulmonary infectious diseases was used to prepare bronchoalveolar lavage cell(BALC). The ratio of MDSC to PBMC, and the ratio of MDSC to BALC was detected by flow cytometry respectively. The same method was used to detect the ratio of MDSC to PBMC among active PTB patients after therapy,cured PTB patients and recovery PTB patients. PBMC isolated from healthy controls was stained with CFSE living cell fluorescence dyeing agent( CFSE - PBMC),CD +11b CD -14 MDSC was isolated from PBMC in active PTB patients,and PBMC without MDSC was treated with mitomycin C. 2 × 105 CFSE - PBMC were used as reaction cells,2 × 105 PBMC without MDSC and 2 × 105 MDSC was mixed and cultured at different ratio(1: 0,1: 2,1: 4,1:8). CFSE signal of CD +3 T cell was determined by using flow cytometry. The serum levels of L - arginine,NO and ornithine in active PTB patients and healthy controls were detected. Results The ratio of MDSC to PBMC among active PTB patients, contacted PTB patients and healthy controls was 0. 338 (0. 274,0. 399 ),0. 115 ( 0. 073,0. 201 )and 0. 042 ( 0. 031, 0. 103),respectively,the difference among the three groups was significant(H = 16. 450,P < 0. 001). The ratio of MDSC to PBMC among active PTB patients was significantly higher than that among healthy controls and that among contacted PTB patients, respectively(P < 0. 001). The ratio of MDSC to BALC among active PTB patients〔0. 425(0. 219,0. 483)〕was significantly higher than that〔0. 031(0. 011,0. 100)〕among pulmonary disease infection patients( Z = 9. 675,P = 0. 004). After PBMC was mixed with MDSC by 1: 0,1: 2,1: 4 and 1: 8,respectively,CD +3 T cell proliferation ratio was 0. 874(0. 761, 0. 953),0. 668( 0. 588,0. 818 ),0. 510 ( 0. 477,0. 614 ) and 0. 186 ( 0. 112,0. 290 ),respectively,the difference among the four groups was significant(H = 9. 995,P = 0. 006). The serum level of L - arginine in active PTB patients〔31. 72 (18. 37,59. 58) μmol/ L〕was significantly lower than that〔57. 88 (35. 11,77. 93) μmol/ L〕in healthy controls( Z= 9. 030,P = 0. 008). The serum level of NO in active PTB patients〔63. 54(51. 83,81. 33) μmol/ L〕was significantly higher than that〔30. 31(28. 19,40. 28)μmol/ L〕in healthy controls(Z = 10. 033,P = 0. 004). After treatment,the ratio of MDSC to PBMC in cured PTB patients〔0. 024(0. 008,0. 0389)〕was significantly lower than that〔0. 323(0. 264, 0. 388)〕in active PTB patients who were under treatment(Z = 18. 880,P < 0. 001). The ratio of MDSC to PBMC in recovery PTB patients〔0. 034(0. 018,0. 039)〕was significantly lower than that〔0. 053(0. 017,0. 084)〕in PTB patients who just finished treatment(Z = 11. 125,P = 0. 004). Conclusion The ratio of MDSC to PBMC and ratio of MDSC to BALC in active PTB patients is high,and these two ratios are markedly low in cured or recovery active PTB patients. MDSC could inhibit T lymphocytes proliferation and it′s mechanism may be related to activated NO signal pathway.