E.g抗原B对体外培养小鼠脾细胞和人PBMC中Treg细胞比例影响的研究

发布时间：2019-05-15 11:49

【摘要】：目的：以BALB/c小鼠脾脏细胞在体外与细粒棘球蚴囊液共培养，简单模拟肝囊型包虫病（CE）患者体内环境，进行前期实验，观察细粒棘球蚴（E.g）囊液对体外培养BABL/c小鼠脾脏细胞中调节性T细胞（Treg细胞）相对特异分子叉头蛋白3（Foxp3）及转化生长因子-β1（TGF-β1）下游信号通路Smad蛋白4（Smad4）表达的影响。进一步试验研究，CE患者和健康志愿者外周血单个核细胞（PBMC）中Treg细胞的比例，以体内和体外实验相比较，观察抗原B在体外对健康志愿者PBMC中Treg细胞的影响，以此来研究抗原B在细粒棘球蚴所致的免疫逃避中对Treg细胞的影响。方法：以BABL/c小鼠作为脾细胞供者，用研磨法分离脾脏细胞，随机分为试验组（与细粒棘球蚴囊液共同培养）和对照组，1×10~5接种于96孔板上，分别在1h，3h，6h，12h提取总RNA，实时荧光定量PCR（qRT-PCR）技术检测Foxp3以及Smad4的基因表达。新疆医科大学第一附属医院25例受试者分为两组：健康对照组(healthy control,HC)10例，肝囊性包虫病（cystic echinococcosis, CE）组15例。Ficoll密度梯度离心法分离PBMC，流式细胞术（flow cytometry, FCM)检测Treg细胞的表达；收集5例健康人外周血，体外分离PBMC，分为阴性对照组，低浓度(终浓度2μg/mL)和高浓度抗原B处理组(终浓度5μg/mL)，分别在96，120和144h收集细胞，流式细胞术检测Treg细胞的表达。采用配对t检验分析组间差异，Pearson相关性检验分析CE病人Treg与AgB的相关性。结果：qRT-PCR结果显示，细粒棘球蚴囊液处理1h Foxp3表达量显著降低（试验组4.577±0.317，对照组9.274±0.451）；3h，12h Foxp3表达量显著升高（试验组8.517±0.978，对照组3.297±0.408；试验组7.406±0.822，对照组2.464±0.328）。细粒棘球蚴囊液处理组1h Smad4表达量显著增加（试验组3.862±1.417，对照组1.689±0.221）；12h Smad4的表达量显著降低（试验组1.690±0.248，对照组3.600±1.081）。并且Foxp3与Smad4二者之间存在负相关，差异有统计学意义（P＜0.05）。流式细胞检测结果显示：与健康对照组（0.800±0.470）比较，肝囊性包虫病组Treg细胞比例（2.540±1.130）显著升高（P＝0.026，P＜0.05）；与阴性对照组比较（0.575±0.126，1.067±0.666），体外培养的正常PBMC在抗原B作用120和144h，低浓度组Treg细胞在PBMC中的比例显著升高（分别为0.800±0.082，2.200±0.819），差异有统计学意义(t＝2.820和t＝2.529，P＜0.01)；高浓度组Treg细胞在PBMC中的比例显著降低（分别为0.333±0.115，0.833±0.551），差异有统计学意义(t＝2.598和t＝2.836，P＜0.05)。且病人血清中抗原B的表达量与Treg细胞呈正相关(r＝0.739，P＜0.05)。结论：细粒棘球蚴囊液上调小鼠脾脏细胞中Treg相对特异分子Foxp3的表达，下调TGF-β1的下游信号通路Smad4的表达，二者之间存在负相关，提示细粒棘球蚴侵染宿主的过程中可能通过负调控TGF-β信号通路而造成宿主Treg细胞的表达升高，进而可能参与细粒棘球蚴感染引起的的免疫逃避。CE病人外周血中Treg细胞表达升高，且与AgB呈正相关。体外实验进一步证实低浓度细粒棘球蚴抗原B可能对正常人外周血单个核细胞中Treg细胞的表达起到促进作用，但随着抗原B浓度的升高，促进作用减弱，，推测抗原B在细粒棘球蚴感染所致的免疫逃避中对Treg细胞分化的起着重要作用。
[Abstract]:Objective: The spleen cells of BALB/ c mice were co-cultured with the fine-grained echinococcin in vitro, and the in-vivo environment of the patients with hepatic cysticercosis (CE) was simply simulated, and the early-stage experiments were carried out. The expression of Smad-4 (Smad4) in the downstream signal pathway of T cell (Tregs) in the spleen of BABL/ c mice and the expression of Smad-4 (Smad4) were observed in the spleen cells of BABL/ c mice. The ratio of Treg cells in peripheral blood mononuclear cells (PBMC) of CE patients and healthy volunteers was further tested, and the effect of antigen B on Treg cells in PBMC of healthy volunteers was observed in vivo and in vitro. In this way, the effect of the antigen B on Treg cells in the immune escape caused by the fine-grained echinococcus is studied. Methods: BABL/ c mice were used as the donor of the spleen cells, and the spleen cells were isolated by the grinding method. The cells were randomly divided into the test group (co-cultured with the fine-grained echinocular capsule) and the control group, and the total RNA was extracted at 1 h,3 h,6 h and 12 h, respectively, and the total RNA was extracted at 1 h,3 h,6 h and 12 h, respectively. The gene expression of Foxp3 and Smad4 was detected by real-time fluorescence quantitative PCR (qRT-PCR). In the first Affiliated Hospital of Xinjiang Medical University,25 subjects were divided into two groups: the health control group (HC) in 10 cases, and the cystic echinococcosis (CE) group in 15 cases. The expression of Treg cells was detected by Ficoll density gradient centrifugation, and the expression of Treg cells was detected by flow cytometry (FCM). The peripheral blood of 5 healthy subjects was collected, and the PBMCs were isolated in vitro, divided into a negative control group, a low concentration (final concentration of 2. mu.g/ mL) and a high concentration antigen B treatment group (final concentration of 5. mu.g/ mL). Cells were collected at 96,120 and 144h, respectively, and the expression of Treg cells was detected by flow cytometry. The correlation between Treg and AgB in the CE patients was analyzed by Pearson correlation test using the paired t-test analysis group differences. Results: The results of qRT-PCR showed that the expression of Foxp3 was significantly reduced by qRT-PCR (4.577-0.317 in the test group and 9.274-0.451 in the control group). The expression of Foxp3 in the 3-h,12-h Foxp3 was significantly higher (8.517-0.978 in the test group, 3.297-0.408 in the control group, 7.406-0.822 in the test group and 2.464-0.328 in the control group). The expression of Smad4 was significantly increased in the treatment group (3.862-1.417 in the test group and 1.689-0.221 in the control group), and the expression of the 12-h Smad4 was significantly decreased (1.690-0.248 in the test group and 3.600-1.081 in the control group). There was a negative correlation between Foxp3 and Smad4 (P <0.05). The results of flow cytometry showed that, compared with the healthy control group (0.800-0.470), the proportion of Treg cells (2.540-1.130) in the hepatic-cystic hydatid disease group was significantly higher (P = 0.026, P <0.05); compared with the negative control group (0.575-0.126, 1.067-0.666), the normal PBMCs were cultured in vitro for 120 and 144h. The proportion of Treg cells in the low-concentration group was significantly higher in PBMCs (0.800, 0.082, 2.200-0.819, respectively), and the difference was statistically significant (t = 2.820 and t = 2.529, P <0.01), and the proportion of Treg cells in the high-concentration group was significantly lower in PBMCs (0.333-0.115, 0.833-0.551, respectively). The difference was statistically significant (t = 2.598 and t = 2.836, P <0.05). And the expression of the antigen B in the serum of the patient was positively correlated with the Treg cell (r = 0.739, P <0.05). Conclusion: The expression of Treg relative to the specific molecular Foxp3 in the spleen cells of the mouse is up-regulated by the fine-grained echinocular capsule, and there is a negative correlation between the expression of the downstream signal pathway Smad4 of the TGF-CD1. It is suggested that the expression of the host Treg cells may be increased by the negative regulation of the TGF-1 signaling pathway in the process of infecting the host, and the immune escape caused by the infection of the fine-grained echinococcus may be involved. The expression of Treg cells in peripheral blood of CE patients was elevated and was positively correlated with AgB. in vitro, it is further confirmed that the low-concentration fine-grained echinocandin antigen B can promote the expression of Treg cells in the peripheral blood mononuclear cells of a normal person, but with the increase of the concentration of the antigen B, the promoting effect is weakened, It is assumed that the antigen B plays an important role in the differentiation of Treg cells in the immune escape caused by the infection of the fine-grained echinococcus.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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