肺结核患者外周血Tγδ17细胞的亚群分析以及结核杆菌抗原对诱导Tγδ17细胞分化的影响

发布时间：2018-09-16 20:22

【摘要】：研究背景：在宿主对结核分枝杆菌（Mtb）感染的免疫机制中，γδ T细胞的作用越来越受到关注和重视。近年研究报道Mtb和卡介苗感染的小鼠模型中分泌IL-17的细胞主要来源于γδ T细胞。本研究室近期的研究也发现活动性肺结核（TB）患者外周血中产生IL-17的γδ T细胞（Tγδ17细胞）的比例明显高于正常健康人。但Tγδ17细胞是属于γδ T细胞的何种亚群，以及肺TB患者的Tγδ17细胞的亚群类别与健康人有何不同，均尚未见报道。另外，虽然对于诱导Th17细胞分化的关键性细胞因子和转录因子已经明确，但是对于诱导Tγδ17细胞分化的细胞因子，转录因子和抗原的调节作用等影响因素的研究很少，尚有待探讨阐明。目的：检测TB患者和健康人（HD）外周血中Tγδ17细胞属于γδ T细胞何种亚群（Vδ1、Vδ2及Vδ3-8亚群）探讨肺TB患者的Tγδ17细胞的亚群类别与健康人有何不同,以及结核杆菌（M.tb）耐热抗原（Mtb-HAg）、磷酸化抗原（HDMAPP）加入细胞因子诱导Tγδ17细胞分化有何不同，以探讨不同淋巴细胞亚群在抗M.tb感染免疫应答和TB发病机制中的作用。方法：1.采集70例活动性肺结核病人、100例HD和33例矽肺患者外周血，加入荧光标记单抗进行表面分子染色，流式细胞仪检测γδT细胞亚群（Vδ1、Vδ2及Vδ3-8亚群）的比例变化。2.采集24例活动性肺结核病人和17例HD外周血，加入佛波醇酯（PMA）和钙离子霉素（Ionomycin）刺激培养2小时后，再加入莫能霉素（Monensin）继续培养4小时，收集细胞，加入荧光标记单抗进行表面分子和胞内染色，流式细胞仪检测产生Tγδ17细胞亚群（Vδ1、Vδ2及Vδ3-8亚群）比例及。3.采用密度梯度离心法分离外周血单个核细胞（PBMC），加入Mtb-Hag、HDMAPP和细胞因子（IL-1β、IL-23和TGF-β）培养3天后，加入rIL-2刺激培养9天后，使其成为富含效应性γδ T细胞的细胞群，收集细胞用PMA/Ionomycin和Monensin刺激培养6小时，，用流式细胞仪检测Tγδ17细胞亚群的比例。结果： 1、TB患者60岁初治组（n＝16）γδ T细胞在T细胞中的比例（5.26%）低于正常人60岁组（n＝23）的比例（6.3%），TB患者60岁复治组（n＝16）γδ T细胞在T细胞中的绝对值（74.52%）低于TB患者60岁初治组60岁组（n＝34）的绝对值（144.69）（绝对值单位为101/μl）。 2、TB患者60岁初治组（n＝32）复治组（n＝16）的Vδ2+亚群在总γδ T细胞中比例（60.06%和48.82%）明显低于正常人60岁组（n＝75）的比例（73.68%）（p均0.01）。TB患者60岁初治组（n＝32）复治组（n＝11）的Vδ1+亚群在总γδ T细胞中比例（25.01%和22.61%）明显高于正常人60岁组（n＝75）的比例（11.82%）（p均0.05）。TB患者60岁复治组（n＝14）的Vδ3-8+亚群在总γδ T细胞中比例（33.17%）明显高于正常人60岁组（n＝21）的比例（14.49%）（p0.05）。TB患者60岁初治组（n＝14）的Vδ1+亚群在总γδ T细胞中的绝对值（3.71）明显低于TB患者60岁复治组（n＝10）的绝对值（11.71）。TB患者60岁初治组（n＝14）的Vδ3-8+亚群在总γδ T细胞中的绝对值（3.03）明显低于TB患者60岁初治组（n＝32）的绝对值（14.89）（p均0.05.单位为101/μl）。 3、结核病组Tγδ17细胞中以Vδ3-8亚群最多，其次为Vδ2亚群，最少为Vδ1亚群。正常组Tγδ17细胞以Vδ3-8亚群最多，其次为Vδ1亚群，最少为Vδ2亚群。其中正常组γδ T细胞亚群:Vδ1亚群（n=14）、Vδ2亚群（n=18）和Vδ3-8亚群（n=14）的百分比分别为27.31%、10.52%和63.46%，而结核病组γδ T细胞亚群:Vδ1亚群（n=23）、Vδ2亚群（n=24）和Vδ3-8亚群（n=23）的百分比分别为5.03%、10.52%和75.03%。 4、从γδT细胞亚群的绝对值上进行比较，正常组中产生Tγδ17细胞的亚群类别中Vδ1亚群与Vδ3-8亚群绝对值相当，而结核病组产生Tγδ17细胞的亚群类别Vδ3-8亚群绝对值急剧升高。其中正常组γδ T细胞亚群:Vδ1亚群（n=14）、Vδ2亚群（n=18）和Vδ3-8亚群（n=14）的绝对值分别为2.46、0.19和2.78，而结核病组γδ T细胞亚群:Vδ1亚群（n=23）、Vδ2亚群（n=24）和Vδ3-8亚群（n=23）的绝对值分别为0.64、0.46和3.7。（单位为101/μl） 5、Mtb-Ag+CK组与HDMAPP+CK组在Tγδ17的比例比较中有明显差异，前一组明显比后组升高。Mtb-Ag+CK组与HDMAPP+CK组中Vδ2在Tγδ17所占比例比较中，前者较后者明显降低。结论： 1、在正常人中γδ T细胞主要以Vδ2+亚群为主，而在初治结核病、复治结核病以及矽肺患者中，Vδ2+亚群比例降低，而Vδ1+亚群比例增高或Vδ3-8+亚群比例增高。 2、TB患者60岁初治组复治组的Vδ2+亚群在总γδ T细胞中比例明显低于正常人60岁组的比例。TB患者60岁初治组复治组的Vδ1+亚群在总γδ T细胞中比例明显高于正常人60岁组的比例。TB患者60岁复治组的Vδ3-8+亚群在总γδ T细胞中比例明显高于正常人60岁组的比例。 3、在正常人及TB患者，Tγδ17细胞中均以Vδ3-8亚群最多（60％以上）；正常人Tγδ17细胞中Vδ2亚群最少（半数人群检测不到），而TB患者中的Tγδ17细胞中Vδ2亚群明显升高；TB患者的Tγδ17细胞中Vδ1亚群明显低于正常人。 4、正常人PBMC，用Mtb-HAg和磷酸抗原（HDMAPP）激活γδT细胞并同时加入含诱导性细胞因子（IL-1β、IL-23和TGF-β）培养（12天）后，均可诱导Tγδ17细胞的分化和增殖。前者诱导产生Tγδ17细胞的数量明显多于后者。但在前者诱导的Tγδ17细胞中，Vδ2细胞数量明显低于后者诱导的Tγδ17。
[Abstract]:BACKGROUND: The role of gamma delta T cells in host immunity against Mycobacterium tuberculosis (Mtb) infection has attracted more and more attention. Recent studies have reported that IL-17 secreting cells in mice infected with Mtb and BCG are mainly derived from gamma delta T cells. Recent studies in this laboratory have also found that the cells secreting IL-17 mainly come from gamma delta T cells outside the patients with active pulmonary tuberculosis (TB). The percentage of interleukin-17 (IL-17) producing gamma delta T cells (Tgamma delta 17 cells) in peripheral blood was significantly higher than that in normal controls. However, what subsets of T-gamma delta 17 cells belong to, and how the subtypes of T-gamma delta 17 cells in patients with pulmonary TB differ from those in healthy controls have not been reported. And transcription factors have been identified, but few studies have been done on the factors influencing the differentiation of T gamma delta 17 cells, such as cytokines, transcription factors and antigen regulation.
Objective: To investigate the subtypes of T gamma delta 17 cells (Vdel 1, Vdel 2 and Vdel 3-8) in peripheral blood of TB patients and healthy persons (HD) and to explore the differences between Tgamma delta 17 cells in TB patients and healthy persons, and the effects of thermostable antigen (M tb-HAg) of Mycobacterium tuberculosis (M.tb) and phosphorylated antigen (HDMAPP) on Tgamma delta 17 cells induced by cytokines. To explore the role of different lymphocyte subsets in the immune response to M.tb infection and the pathogenesis of TB.
Methods: 1. The peripheral blood samples of 70 active pulmonary tuberculosis patients, 100 HD patients and 33 silicosis patients were stained with fluorescent labeled monoclonal antibodies. The ratio of gamma delta T lymphocyte subsets (Vdel 1, Vdel 2 and Vdel 3-8) was detected by flow cytometry. 2. 24 active pulmonary tuberculosis patients and 17 HD patients were collected and the peripheral blood samples were added with phorbol alcohol ester (PMA) and phorbol alcohol ester (Vdel 3-8). Calcium ionomycin (Ionomycin) stimulated culture for 2 hours, then added monensin to culture for 4 hours. Cells were collected and labeled with fluorescent monoclonal antibodies for surface molecular and intracellular staining. Flow cytometry was used to detect the proportion of T gamma delta 17 cell subsets (Vdelta 1, Vdelta 2 and Vdelta 3-8) and. 3. Peripheral blood was isolated by density gradient centrifugation. Mononuclear cells (PBMC) were cultured with Mtb-Hag, HDMAPP and cytokines (IL-1 beta, IL-23 and TGF-beta) for 3 days, then stimulated with rIL-2 for 9 days. The collected cells were cultured with PMA/Ionomycin and Monosin for 6 hours. The ratio of T-gamma delta 17 subsets was detected by flow cytometry.
Result:
1. The percentage of gamma delta T cells in T cells in the 60-year-old newly treated group (n=16) was lower than that in the 60-year-old normal group (6.3%). The absolute value of gamma delta T cells (74.52%) in the 60-year-old retreated group (n=16) was lower than that in the 60-year-old newly treated group (n=34) of TB patients (144.69) (the absolute value unit was 101/ml).
2. The proportion of Vdelta 2+ subsets in total gamma delta T cells (60.06% and 48.82%) in the retreated group (n=16) of TB patients aged 60 years old was significantly lower than that in the normal group (73.68%) (p 0.01). The proportion of Vdelta 1+ subsets in total gamma delta T cells (25.01% and 22.61%) in the retreated group of TB patients aged 60 years old (n=32) was significantly higher than that in the normal group (n=11). The proportion of Vdelta 3-8 + subsets in total gamma delta T cells (33.17%) of TB patients aged 60 years after retreatment (n = 14) was significantly higher than that of normal people aged 60 years (n = 21) (14.49%) (p0.05). The absolute value of Vdelta 1 + subsets in total gamma delta T cells (3.71) of TB patients aged 60 years after retreatment was significantly lower than that of TB patients aged 60 years after retreatment (n = 14). The absolute value (11.71) of the treatment group (n = 10). The absolute value (3.03) of the Vdelta 3-8 + subsets in the total gamma delta T cells in the newly treated group (n = 14) of TB patients aged 60 was significantly lower than that in the newly treated group (n = 32) (14.89) (p 0.05.
3. The Vdelta 3-8 subset was the most common in Tgamma delta 17 cells of tuberculosis group, followed by Vdelta 2 subset and Vdelta 1 subset at least. The Vdelta 3-8 subset was the most common in Tgamma delta 17 cells of normal group, followed by Vdelta 1 subset and Vdelta 2 subset at least. The percentages of gamma delta T cell subsets in tuberculosis group were 5.03%, 10.52% and 75.03%, respectively.
4. Comparing the absolute values of the subtypes of T_ Delta T cells, the absolute values of V_ delta 1 and V_ delta 3-8 in the subtypes of T_ delta 17 cells in the normal group were the same, while the absolute values of V_ delta 3-8 in the subtypes of T_ delta 17 cells in the tuberculosis group increased sharply. The absolute values of the subgroup (n = 14) were 2.46, 0.19 and 2.78 respectively, while those of the gamma delta T cell subgroup (n = 23), V delta 2 (n = 24) and V delta 3-8 (n = 23) in the tuberculosis group were 0.64, 0.46 and 3.7, respectively.
5. The ratio of T gamma delta 17 in Mtb-Ag+CK group was significantly higher than that in HDMAPP+CK group. The ratio of V delta 2 in Mtb-Ag+CK group was significantly lower than that in HDMAPP+CK group.
Conclusion:
1. In normal subjects, Vdelta 2+ subsets were predominant in gamma delta T cells, while Vdelta 2+ subsets were decreased in patients with newly treated tuberculosis, retreated tuberculosis and silicosis, and Vdelta 1+ subsets were increased or Vdelta 3-8+ subsets were increased.
2. The proportion of Vdelta 2 + subsets in total gamma delta T cells in the retreated group of TB patients aged 60 was significantly lower than that in the normal group aged 60. The proportion of Vdelta 1 + subsets in total gamma delta T cells in the retreated group aged 60 was significantly higher than that in the normal group aged 60. The proportion of people aged 60 years old.
3. In normal and TB patients, the Vdelta 3-8 subset was the most common (more than 60%) in Tgam delta 17 cells, the Vdelta 2 subset was the least in normal Tgam delta 17 cells (not detected in half of the population), and the Vdelta 2 subset in Tgam delta 17 cells was significantly higher in TB patients than in normal subjects.
4. Normal PBMC, activated by Mtb-HAg and phosphoric acid antigen (HDMAPP) and cultured with inducible cytokines (IL-1 beta, IL-23 and TGF-beta) for 12 days, could induce the differentiation and proliferation of T gamma delta 17 cells. The number of T gamma delta 17 cells induced by the former was significantly higher than that by the latter. The amount of T gamma delta 17. is significantly lower than that of the latter.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R521

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