本文選題：日本血吸蟲 + 抗獨(dú)特型抗體��； 參考：《南京醫(yī)科大學(xué)》2012年碩士論文

【摘要】：日本血吸蟲單克隆抗獨(dú)特型抗體NP30為腸相關(guān)抗原(GAA)的內(nèi)影像抗獨(dú)特型抗體,具有模擬抗原的作用,可替代蟲源抗原用于血吸蟲病免疫診斷和疫苗的研究應(yīng)用NP30主動(dòng)免疫昆明種小鼠、C57BL/6、BALB/c小鼠和山羊,對(duì)尾蚴攻擊感染分別可誘導(dǎo)50.46%、42.05%、39.53%和42.86%[1]的保護(hù)力。 Thl7細(xì)胞是一種新發(fā)現(xiàn)的CD4-T細(xì)胞亞群。研究表明,Th17細(xì)胞通過其主要表達(dá)產(chǎn)物IL-17在血吸蟲感染免疫中發(fā)揮重要作用[2-4]。在曼氏血吸蟲攻擊宿主的早期,Th17被激活并大量分泌IL-17, IL-17可刺激多種分泌各種炎癥相關(guān)細(xì)胞因子；感染情況嚴(yán)重同時(shí)缺乏針對(duì)性治療或宿主免疫功能低下時(shí),血吸蟲蟲荷增加,誘導(dǎo)更多的Th17細(xì)胞分化,對(duì)宿主產(chǎn)生嚴(yán)重的免疫病理改變[5-7]；在曼氏血吸蟲感染后期,Th17細(xì)胞及其分泌的IL-17還參與肝臟蟲卵肉芽腫形成[7],這提示蟲卵導(dǎo)致的免疫病理?yè)p傷很大程度上是由Thl7細(xì)胞介導(dǎo)。由此推測(cè)Th17在日本血吸蟲感染宿主免疫中也起了重要作用。 目的 鑒于IL-17與血吸蟲感染關(guān)系密切,推測(cè)NP30誘導(dǎo)的宿主保護(hù)性免疫與Th17細(xì)胞分泌的IL-17有關(guān),引出以下研究目的： 1、闡明Th17與日本血吸蟲單克隆抗獨(dú)特型抗體NP30誘導(dǎo)宿主保護(hù)性免疫的關(guān)系； 2、為進(jìn)一步提高日本血吸蟲單克隆抗獨(dú)特型抗體NP30的免疫保護(hù)作用提供理論依據(jù)。 方法 1、小鼠骨髓源性樹突狀細(xì)胞(DCs)的體外培養(yǎng)并鑒定 取正常BALB/c小鼠骨髓源細(xì)胞,利用GM-CSF(10ng/ml)及IL-4(5ng/ml)誘導(dǎo)培養(yǎng)DCs,倒置顯微鏡下觀察其形態(tài),流式細(xì)胞術(shù)檢測(cè)其表面標(biāo)志。 2、體外檢測(cè)NP30刺激后IFN-γ、IL-4、IL-6、IL-17、IL-23和TGF-β水平的變化 取正常BALB/c小鼠脾淋巴細(xì)胞,分選CD4+T細(xì)胞,不同濃度NP30、SEA與DCs+CD4+T細(xì)胞共培養(yǎng),對(duì)照組用PBS。培養(yǎng)72h后收集細(xì)胞上清,檢測(cè)各組分泌上述細(xì)胞因子的水平,初步篩選NP30相關(guān)的細(xì)胞因子。 3、檢測(cè)NP30免疫后感染小鼠骨髓源DCs以及CD4+T細(xì)胞中與Th17細(xì)胞分化相關(guān)的因子水平 取6周齡BALB/c小鼠30只,每組10只,分為NP30組(用NP30免3次后感染日本血吸蟲尾蚴40±±2條)；SEA組(用SEA免疫3次后感染日本血吸蟲尾蚴40±-2條)；對(duì)照組(注射PBS后感染日本血吸蟲尾蚴40±±2條),分別在感染5w、8w后取骨髓源DCs以及脾細(xì)胞,不同抗原刺激后,檢測(cè)細(xì)胞因子IFN-y、IL-4、IL-6、IL-17、IL-23和TGF-β的變化。 結(jié)果 1、從小鼠骨髓細(xì)胞中誘導(dǎo)培養(yǎng)出可供實(shí)驗(yàn)用的DCs,純度為87.37%,經(jīng)NP30刺激,DCs共刺激分子CD86有所增高； 2、體外實(shí)驗(yàn)顯示：NP30刺激DCs分泌較高水平的TGF-p,而IL-6和IL-23水平明顯低于SEA組；NP30刺激DCs+CD4+T細(xì)胞分泌IL-17及與Th17細(xì)胞分化相關(guān)因子水平雖高于PBS組,但明顯低于SEA組； 3、體內(nèi)實(shí)驗(yàn)表明：感染后5w NP30免疫組DCs分泌IL-6、TGF-β水平低于SEA免疫組；脾臟來源的CD4+T細(xì)胞在NP30刺激下,TGF-β水平高于SEA免疫組,而IL-17表達(dá)較SEA免疫組有所降低。感染后8w NP30免疫組CD4+T細(xì)胞IL-17水平明顯亦低于其他各組。 結(jié)論 1、Th17細(xì)胞在日本血吸蟲感染免疫中具有重要作用； 2、日本血吸蟲單克隆抗獨(dú)特型抗體NP30誘導(dǎo)宿主保護(hù)性免疫與調(diào)節(jié)Th17反應(yīng)相關(guān)； 3、可能通過以下兩種機(jī)制：①改變DCs分泌Thl7細(xì)胞相關(guān)因子的水平類型；②促進(jìn)Treg細(xì)胞分化調(diào)節(jié)Thl7極化。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 郝陽(yáng);鄭浩;朱蓉;郭家鋼;王立英;陳朝;周曉農(nóng);;2009年全國(guó)血吸蟲病疫情通報(bào)[J];中國(guó)血吸蟲病防治雜志;2010年06期

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