Th17细胞在NP30诱导宿主保护性免疫中的作用

发布时间：2018-04-12 15:06

本文选题：日本血吸虫 + 抗独特型抗体　；参考：《南京医科大学》2012年硕士论文

【摘要】：日本血吸虫单克隆抗独特型抗体NP30为肠相关抗原(GAA)的内影像抗独特型抗体,具有模拟抗原的作用,可替代虫源抗原用于血吸虫病免疫诊断和疫苗的研究应用NP30主动免疫昆明种小鼠、C57BL/6、BALB/c小鼠和山羊,对尾蚴攻击感染分别可诱导50.46%、42.05%、39.53%和42.86%[1]的保护力。 Thl7细胞是一种新发现的CD4-T细胞亚群。研究表明,Th17细胞通过其主要表达产物IL-17在血吸虫感染免疫中发挥重要作用[2-4]。在曼氏血吸虫攻击宿主的早期,Th17被激活并大量分泌IL-17, IL-17可刺激多种分泌各种炎症相关细胞因子；感染情况严重同时缺乏针对性治疗或宿主免疫功能低下时,血吸虫虫荷增加,诱导更多的Th17细胞分化,对宿主产生严重的免疫病理改变[5-7]；在曼氏血吸虫感染后期,Th17细胞及其分泌的IL-17还参与肝脏虫卵肉芽肿形成[7],这提示虫卵导致的免疫病理损伤很大程度上是由Thl7细胞介导。由此推测Th17在日本血吸虫感染宿主免疫中也起了重要作用。目的鉴于IL-17与血吸虫感染关系密切,推测NP30诱导的宿主保护性免疫与Th17细胞分泌的IL-17有关,引出以下研究目的： 1、阐明Th17与日本血吸虫单克隆抗独特型抗体NP30诱导宿主保护性免疫的关系； 2、为进一步提高日本血吸虫单克隆抗独特型抗体NP30的免疫保护作用提供理论依据。方法 1、小鼠骨髓源性树突状细胞(DCs)的体外培养并鉴定取正常BALB/c小鼠骨髓源细胞,利用GM-CSF(10ng/ml)及IL-4(5ng/ml)诱导培养DCs,倒置显微镜下观察其形态,流式细胞术检测其表面标志。 2、体外检测NP30刺激后IFN-γ、IL-4、IL-6、IL-17、IL-23和TGF-β水平的变化取正常BALB/c小鼠脾淋巴细胞,分选CD4+T细胞,不同浓度NP30、SEA与DCs+CD4+T细胞共培养,对照组用PBS。培养72h后收集细胞上清,检测各组分泌上述细胞因子的水平,初步筛选NP30相关的细胞因子。 3、检测NP30免疫后感染小鼠骨髓源DCs以及CD4+T细胞中与Th17细胞分化相关的因子水平取6周龄BALB/c小鼠30只,每组10只,分为NP30组(用NP30免3次后感染日本血吸虫尾蚴40±±2条)；SEA组(用SEA免疫3次后感染日本血吸虫尾蚴40±-2条)；对照组(注射PBS后感染日本血吸虫尾蚴40±±2条),分别在感染5w、8w后取骨髓源DCs以及脾细胞,不同抗原刺激后,检测细胞因子IFN-y、IL-4、IL-6、IL-17、IL-23和TGF-β的变化。结果 1、从小鼠骨髓细胞中诱导培养出可供实验用的DCs,纯度为87.37%,经NP30刺激,DCs共刺激分子CD86有所增高； 2、体外实验显示：NP30刺激DCs分泌较高水平的TGF-p,而IL-6和IL-23水平明显低于SEA组；NP30刺激DCs+CD4+T细胞分泌IL-17及与Th17细胞分化相关因子水平虽高于PBS组,但明显低于SEA组； 3、体内实验表明：感染后5w NP30免疫组DCs分泌IL-6、TGF-β水平低于SEA免疫组；脾脏来源的CD4+T细胞在NP30刺激下,TGF-β水平高于SEA免疫组,而IL-17表达较SEA免疫组有所降低。感染后8w NP30免疫组CD4+T细胞IL-17水平明显亦低于其他各组。结论 1、Th17细胞在日本血吸虫感染免疫中具有重要作用； 2、日本血吸虫单克隆抗独特型抗体NP30诱导宿主保护性免疫与调节Th17反应相关； 3、可能通过以下两种机制：①改变DCs分泌Thl7细胞相关因子的水平类型；②促进Treg细胞分化调节Thl7极化。
[Abstract]:Schistosoma japonicum monoclonal anti idiotypic antibody NP30 of gut associated antigen (GAA) of the internal image anti idiotypic antibody, has simulated the role of antigen, can replace the insect source for active immune antigen diagnosis and vaccine of schistosomiasis research and application of NP30 Kunming mice, C57BL/6 BALB/c mice and goats of cercariae infection can induce respectively. 50.46%, 42.05%, 39.53% protection and 42.86%[1].
Thl7 is a newly discovered cell subpopulation of CD4-T cells. The results showed that Th17 cells through its main expression product IL-17 in Schistosoma japonicum infection play an important role in the early stage of [2-4]. of Schistosoma mansoni host immune attack, Th17 was activated and secrete large amounts of IL-17, IL-17 can stimulate the secretion of many various inflammatory cytokines; severe infection at the same time, the lack of targeted therapy or host immune function, Schistosoma parasite load increases, more Th17 induced cell differentiation, immune pathological changes serious [5-7] on host; Schistosoma mansoni infection, Th17 cells and the secretion of IL-17 in granuloma formation [7], suggesting that the immune pathological damage caused by large eggs the degree is mediated by Thl7 cells. The result suggested that Th17 host immunity also plays an important role in the infection of Schistosoma japonicum.
objective
In view of the close relationship between IL-17 and Schistosoma infection, it is speculated that the host protective immunity induced by NP30 is related to the IL-17 secreted by Th17 cells, which leads to the following research purposes:
1, to elucidate the relationship between Th17 and the protective immunity induced by monoclonal anti idiotypic antibody NP30 of Schistosoma japonicum.
2, it provides a theoretical basis for further improving the protective effect of the monoclonal anti idiotypic antibody NP30 of Schistosoma japonicum.
Method
1, in vitro culture and identification of mouse bone marrow derived dendritic cells (DCs) in vitro
Bone marrow cells from normal BALB/c mice were cultured, and DCs was induced by GM-CSF (10ng/ml) and IL-4 (5ng/ml). Morphology was observed under inverted microscope, and surface markers were detected by flow cytometry.
2, changes in levels of IFN- gamma, IL-4, IL-6, IL-17, IL-23 and TGF- beta after NP30 stimulation in vitro
Normal BALB/c mice spleen lymphocytes were selected, CD4+T cells were sorted, NP30 and SEA were co cultured with DCs+CD4+T cells, the control group was cultured with 72h after PBS., the cell supernatants were collected, the levels of cytokines secreted above were detected, and NP30 related cytokines were screened preliminarily.
3, detection of factors associated with Th17 cell differentiation in NP30 infected mice bone marrow source DCs and CD4+T cells
30 6 week old BALB/c mice, 10 rats in each group were divided into NP30 group (with NP30 from cercariae of Schistosoma japonicum infection after 3 + 40 + 2); SEA group (immunized with SEA after 3 times of infection of Schistosoma japonicum cercariae 40 + -2); control group (injection of PBS after infected by Schistosoma japonicum 40 + + 2, respectively) were infected with 5W, 8W after DCs from bone marrow and spleen cells, different antigen stimulated cytokine detection, IFN-y, IL-4, IL-6, IL-17, IL-23 changes and TGF- beta.
Result
1, DCs from mouse bone marrow cells was induced and cultured for experimental use. The purity was 87.37%. After NP30 stimulation, the DCs co stimulator CD86 increased.
2, in vitro experiments showed that NP30 stimulated DCs to secrete high level TGF-p, while IL-6 and IL-23 levels were significantly lower than those in SEA group. NP30 stimulated DCs+CD4+T cells to secrete IL-17 and Th17 related differentiation factor levels were higher than those in the PBS group, but significantly lower than those in the IL-17 group.
3 in vivo experiments showed that IL-6 5W NP30 immune group DCs secretion after infection, TGF- beta level is lower than the SEA group; spleen derived CD4+T cells under NP30 stimulation, TGF- beta level is higher than SEA immune group, while the expression of IL-17 SEA immune group decreased. After infection of CD4+T cells IL-17 8W NP30 group was also immune level lower than the other groups.
conclusion
1, Th17 cells play an important role in the immunization of Schistosoma japonicum.
2, the monoclonal anti idiotypic antibody NP30 of Schistosoma japonicum is related to the protective immunity of the host and the regulation of the Th17 response.
3, the following two mechanisms are possible: (1) changes in the level of DCs secreting Thl7 cell related factors; (2) promoting the differentiation of Treg cells to regulate the polarization of Thl7.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】