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目的探讨约氏疟原虫(Plasmodium yoelii)17XL株(Py17XL)感染小鼠过程中树突状细胞(DCs)对辅助性T细胞17型(Thl7)细胞分化和功能的调控作用及机制。方法将12只雌性BALB/c小鼠随机分为感染组(Py17XL)、Toll样受体4(TLR4)阻断组(Py17XL+TLR4)、Toll样受体9(TLR9)阻断组(Py17XL+TLR9)和TLR4联合TLR9阻断组(Py17XL+TLR4+TLR9),每组3只。感染前1 d,腹腔注射10μg抗-TLR4单抗(TLR4阻断组)或50μg抗-TLR9单抗(TLR9阻断组)阻断DCs的功能,注射体积为0.4 ml,对照组注射等量的PBS。4组小鼠均腹腔注射1×10~6 Py17XL感染的红细胞。感染后第0、3和5天计数红细胞感染率,同时制备脾细胞悬液,流式细胞仪检测脾脏细胞中CD11c+TLR9+和Th17细胞数量变化,ELISA检测脾细胞培养上清中干扰素-γ(IFN-γ)和白细胞介素-10(IL-10)水平的变化。结果流式细胞仪检测表明,DCs阻断模型构建成功。感染后第5天,感染组、TLR4阻断组、TLR9阻断组和TLR4联合TLR9阻断组小鼠原虫血症分别为28.0%、29.0%、31.0%和16.3%;感染后第7天,原虫血症分别为43.3%、47.5%、32.5%和8.0%,感染组和TLR4阻断组小鼠全部死亡,其余阻断组小鼠于感染后第6天开始出现死亡。与感染组比较,TLR9阻断组和TLR4联合TLR9阻断组小鼠原虫血症上升缓慢,生存期明显延长至11 d和15 d。流式细胞仪检测结果显示:感染后第5天,TLR9阻断组和TLR4联合TLR9阻断组Th17细胞数量分别为1.2%和1.4%,与感染组(1.9%)相比,数量明显减少(P<0.05,P<0.01)。ELISA检测结果显示:感染后第5天,TLR4联合TLR9阻断组小鼠IFN-γ和IL-10水平分别为(232.4±15.5)pg/ml和(1 791.2±58.2)pg/ml,与感染组[(90.7±50.1)pg/ml和(962.6±409.0)pg/ml]相比,差异有统计学意义(P<0.05,P<0.01)。结论 Py17XL感染小鼠过程中DCs调控了Thl7细胞的分化和功能。DCs阻断(Py17XL+TLR4+TLR9)后,感染小鼠原虫血症降低,生存期延长。
Objective To investigate the regulatory effect of dendritic cells (DCs) on the differentiation and function of T helper 17 (Th17) cells in mice infected with Plasmodium yoelii strain 17XL (Py17XL). Methods Twelve female BALB / c mice were randomly divided into four groups: Py17XL, Py17XL + TLR4, Py17XL + Toll-like receptor 9 (TLR9) TLR9) and TLR4 combined with TLR9 blocking group (Py17XL + TLR4 + TLR9), three in each group. One day before infection, the function of DCs was blocked by intraperitoneal injection of 10μg anti-TLR4 monoclonal antibody (TLR4 blocking group) or 50μg anti-TLR9 monoclonal antibody (TLR9 blocking group). The injection volume was 0.4 ml. PBS. 4 groups of mice were injected intraperitoneally 1 × 10 ~ 6 Py17XL infected red blood cells. The infection rate of erythrocytes was counted on the 0, 3 and 5 days after infection, and the spleen cell suspension was prepared. The numbers of CD11c + TLR9 + and Th17 cells in spleen cells were detected by flow cytometry. The levels of IFN- (IFN-γ) and interleukin-10 (IL-10) levels. Results Flow cytometry showed that DCs block model was successfully constructed. On the fifth day after infection, parasitemia in the infection group, TLR4 blocking group, TLR9 blocking group and TLR4 combined with TLR9 blocking group were 28.0%, 29.0%, 31.0% and 16.3% respectively. On the 7th day after infection, Parasitemia was 43.3%, 47.5%, 32.5% and 8.0%, respectively. All the mice in the infected group and the TLR4-blocking group died, and the other mice in the blocking group started to die on the 6th day after infection. Compared with the infected group, the parasitemia in the TLR9-blocking group and the TLR4-TLR9-blocking group increased slowly and the survival time was significantly prolonged to 11 and 15 days. The results of flow cytometry showed that the number of Th17 cells in TLR9 blocking group, TLR4 blocking group and TLR9 blocking group were 1.2% and 1.4% on the 5th day after infection, respectively, which were significantly decreased compared with the infected group (1.9%) P <0.05, P <0.01). The results of ELISA showed that the levels of IFN-γ and IL-10 in the TLR4 combined with TLR9-blocking group were (232.4 ± 15.5) pg / ml and (1 791.2 ± 58.2) pg / ml, respectively, (P <0.05, P <0.01) compared with the control group [(90.7 ± 50.1) pg / ml and (962.6 ± 409.0) pg / ml] Conclusions DCs in Py17XL-infected mice regulate the differentiation and function of Th17 cells. After DCs block (Py17XL + TLR4 + TLR9), protozoospermia in infected mice decreased and their survival time prolonged.