本文選題：日本血吸蟲 + 童蟲細(xì)胞型免疫原　； 參考：《中南大學(xué)》2008年碩士論文

【摘要】： 第一章Sj童蟲細(xì)胞型與非細(xì)胞型免疫原誘生的保護(hù)性差異 【目的】前期在小鼠模型中研究證明日本血吸蟲(Sj)童蟲活細(xì)胞可誘生抗攻擊感染免疫力。本研究為證明其保護(hù)性效果是否與免疫原物理性狀有關(guān),比較童蟲細(xì)胞型和非細(xì)胞型免疫原所誘生的保護(hù)性差異。【方法】用家兔經(jīng)Sj尾蚴感染后第18d獲得童蟲,剪切法制備童蟲細(xì)胞,細(xì)胞超聲凍融制備非細(xì)胞抗原。動物實驗:取50只單性清潔級昆明鼠隨機(jī)分成對照(PBS)組、童蟲細(xì)胞型抗原(SCA)免疫組和童蟲細(xì)胞非細(xì)胞型抗原(SNCA)免疫組、童蟲碎片抗原(SWFA)免疫組和童蟲可溶性抗原(SWSA)免疫組。經(jīng)小鼠腹股溝皮下肌肉接種免疫原3次,每次間隔2周。末次接種后第4周作攻擊感染(30±1尾/鼠),感染后第45d觀察蟲荷、肝卵荷、肝蟲卵肉芽腫大小。此外,用ELISA檢測實驗鼠血清中特異性抗體動態(tài)變化。【結(jié)果】PCR鑒定所用童蟲細(xì)胞無兔源性成分。保護(hù)性指標(biāo)比較顯示:SCA組、SNCA組、SWFA組和SWSA組的減蟲率分別為46.8%、35.3%、31.4%和8.0%;減卵率分別為:56.2%、38.2%、30.6%和20.2%;SWSA組未見明顯減卵效果(P＞0.05)。SCA組小鼠肝蟲卵肉芽腫顯著小于其他4組(P＜0.05)。免疫學(xué)指標(biāo)檢測顯示:SCA組、SNCA組、SWFA組和SWSA組各鼠血清中均可檢測到抗Sj童蟲抗體,其滴度與免疫次數(shù)呈正相關(guān),在各組間差別不顯著(P＞0.05)�！窘Y(jié)論】進(jìn)一步證明Sj童蟲細(xì)胞型免疫原可誘導(dǎo)小鼠產(chǎn)生較理想的保護(hù)性效果,其誘生機(jī)制之一,與免疫原物理性狀有關(guān)。 第二章Sj童蟲細(xì)胞型與非細(xì)胞型免疫原在小鼠注射部位的動態(tài)變化 【目的】為證明Sj童蟲細(xì)胞型免疫原誘生的保護(hù)性機(jī)制是否與免疫原在宿主體內(nèi)緩慢釋放有關(guān),并觀察細(xì)胞型抗原引起的病理學(xué)變化�！痉椒ā坷ッ魇笤O(shè)A,B兩組:A組經(jīng)大腿皮下肌肉注射10~7童蟲細(xì)胞型抗原;B組注射非細(xì)胞抗原,注后分別在24hrs、3d、6d、9d和12d每組各處理1只小鼠,從注射部位定量獲取組織,用Westernblot法和免疫組化鑒定比較兩種類型抗原消失時間的動態(tài)差異;用組織病理學(xué)觀察炎癥變化動態(tài)。【結(jié)果】SDS-PAGE結(jié)果顯示A、B兩組各組織抗原泳道間的區(qū)帶譜未見明顯差異,Western-blot結(jié)果揭示,A組小鼠組織在細(xì)胞型抗原注射后12d仍可檢測到血吸蟲抗原持續(xù)存在75kDa蛋白條帶,而B組非細(xì)胞型抗原在注后第3d消失。免疫組化檢測抗原的結(jié)果與Western blot結(jié)果相符。A、B兩組小鼠免疫部位的組織病理學(xué)觀察顯示,在注抗原后第1d未見炎性病變,從第3天開始肌肉間隙內(nèi)出現(xiàn)大量炎性細(xì)胞浸潤,其中A組小鼠的炎性病變持續(xù)至抗原注射后12d,但B組消失于抗原注后第12d已基本見不到炎性細(xì)胞浸潤,A、B兩組的炎性細(xì)胞均以中性粒細(xì)胞和淋巴細(xì)胞浸潤為主。兩組鼠的抗原注射部位肌細(xì)胞均未見異常病變�！窘Y(jié)論】血吸蟲童蟲細(xì)胞型免疫原較其非細(xì)胞抗原在接種部位可維持更長時間,具抗原緩釋作用,但引起炎癥病變也較明顯,推測細(xì)胞免疫原的這一特性可能是誘生高保護(hù)性免疫的重要機(jī)制之一。 第三章Sj童蟲細(xì)胞與免疫調(diào)節(jié)劑聯(lián)用的保護(hù)性效果觀察 【目的】觀察免疫調(diào)節(jié)劑可否進(jìn)一步提高Sj童蟲細(xì)胞誘生的免疫保護(hù)性效果。【方法】制備感染后第18d童蟲細(xì)胞。PCR擴(kuò)增鑒定血吸蟲蟲源性細(xì)胞的種屬特異性并檢測細(xì)胞有無宿主成分污染。實驗動物分為8組。A組為PBS對照組;B組為童蟲細(xì)胞免疫組;C組為IL-2注射組;D組為IL-2+童蟲細(xì)胞免疫組;E組為IFN-γ注射組;F組為IFN-γ+童蟲細(xì)胞免疫組;G組為香菇多糖注射組;F組為香菇多糖+童蟲細(xì)胞免疫組。免疫調(diào)節(jié)劑每天注射一次,連續(xù)5d,免疫部位均為雙側(cè)腹股溝皮下肌肉注射,間隔2w 1次,共3次。于末次免疫后第4w采取單盲法對各組小鼠進(jìn)行攻擊感染(30尾/鼠)。感染后第35d剖鼠沖蟲觀察結(jié)果。保護(hù)性效果觀察指標(biāo)包括:計數(shù)蟲荷及肝卵荷;免疫學(xué)觀察指標(biāo)包括:ELISA檢測免疫前后不同時點鼠血清中特異性抗體的動態(tài)變化�！窘Y(jié)果】經(jīng)PCR鑒定證明血吸蟲細(xì)胞免疫原無宿主成分污染。保護(hù)性效果顯示:與A組比較,B組減蟲率為38.4%,減雌率42.1%;C組、D組、E組、F組和G組和H組的減蟲率分別為13.5%、36.1%和32.7%、46.2%、18.3%和33.7%;減雌率分別為-2.1%、21.1%、38.9%、40%、30.5%和40%。B組、C組、D組、E組、F組、G組和H組的LEPG減少率分別為59.8%、26.6%、52.1%、47.7%、57.7%、34%和53.7%;EPF減少率分別為48.6%、25.2%、44.4%、34.7%、50%、28.5%和42.3%。免疫調(diào)節(jié)劑與細(xì)胞聯(lián)合免疫組與單用細(xì)胞組比,特異性抗體水平差異無統(tǒng)計學(xué)意義�！窘Y(jié)論】免疫增強(qiáng)劑均未能顯著提高童蟲細(xì)胞誘生的免疫保護(hù)性效果,但單獨注射IFN-γ有一定的抗日本血吸蟲免疫保護(hù)性效果。
[Abstract]:Chapter one Sj protective differences between cell type and non cellular immunogen of larvae
[Objective] to prove that the living cells of Schistosoma japonicum (Sj) can induce immunity against attack infection in the mouse model. This study is to prove whether the protective effect is related to the physical properties of the immunogen and to compare the protective differences between the cell type and the non cellular immunogen. [Methods] the Sj cercariae of rabbits were used. The child worm was obtained at 18D after dyed, and the cells were prepared by shearing, and the cells were prepared by ultrasonic freezing and thawing. 50 single clean grade Kunming mice were randomly divided into the control group (PBS), the SCA immunization group and the cell non cell antigen (SNCA) immune group, the SWFA immunization group and the children's soluble immune group. SWSA immunization group. The immunogen was inoculated 3 times in the subcutaneous muscle of the mouse, each interval was 2 weeks. The last inoculation was fourth weeks after the last inoculation (30 + 1 tails / rats). After infection, the worm, liver and egg granuloma were observed in 45d. In addition, the dynamic changes of specific antibodies in the serum of the rats were detected by ELISA. [results] PCR identification No rabbit source components were used in children's cells. The protective indexes of SCA, SNCA, SWFA and SWSA were 46.8%, 35.3%, 31.4% and 8%, respectively: 56.2%, 38.2%, 30.6% and 20.2%, respectively, in group SWSA (P > 0.05), the liver egg granuloma in the.SCA group was significantly smaller than the other 4 groups (P < 0.05). Immunology Index detection showed that the anti Sj antibody was detected in the serum of SCA group, SNCA group, SWFA group and SWSA group, and the titer was positively correlated with the number of immune times, and there was no significant difference between each group (P > 0.05). [Conclusion] further proof that the cell type immunogen of Sj can induce a better protective effect and one of the inducing mechanisms. It is related to the physical properties of immunogen.
The second chapter is about the dynamic changes of Sj cell type and non cellular immunogen in injection site of mice.
[Objective] to prove whether the protective mechanism of immunogen induced by Sj cell type is related to the slow release of immunogen in the host and to observe the pathological changes caused by cell type antigen. [Methods] Kunming rats were set up A, B two groups: A group was injected subcutaneous muscle of the thigh to 10~7 cell type antigen of 10~7; group B was injected with non cell antigen and injected after injection. Don't treat 1 mice in each group of 24hrs, 3D, 6D, 9D and 12D, obtain tissue from the injection site, compare the dynamic difference between the two types of antigen disappearance time by Westernblot and immunohistochemistry, and observe the changes of the inflammation by histopathology. [results] SDS-PAGE results show that the band spectrum of A, B two groups of tissue antigens in the B two groups The Western-blot results revealed that 12D in group A mice could still detect the persistence of 75kDa protein bands in 12D after injection of cell antigen, while the non cellular antigens in B group disappeared in post injection 3D. The results of immunohistochemical detection of antigen were in conformity with the results of Western blot, and the histopathology of the immune parts of the mice of B two group was histopathological. The observation showed that there was no inflammatory lesion in 1D after the injection of antigen, and a large number of inflammatory cells began to appear in the intermuscular space from third days. The inflammatory lesions in the A Group continued to 12D after the antigen injection, but the B group disappeared after the antigen injection at 12D, and the inflammatory cells in the group of A and B two were all neutrophils and lymph nodes. There was no abnormal lesion in the myocytes of the two groups. [Conclusion] the cell type immunogen of Schistosoma japonicum can maintain longer than its non cellular antigen at the inoculation site, and has the effect of antigen release, but the inflammatory disease is also more obvious. It is presumed that this characteristic of fine cell immunogen may be the high protection of the inducer. One of the important mechanisms of sexual immunity.
The third chapter is about the protective effect of Sj combined with immunomodulator.
[Objective] to observe whether the immunomodulator could further improve the protective effect of Sj on the cells induced by Sj. [Methods] the species specificity of.PCR amplification and identification of the source cells of Schistosoma japonicum after infection was prepared and detected by the host cell. The experimental animals were divided into 8 groups of.A groups as the PBS control group and the B group as a child. The cell immune group, group C was IL-2 injection group, group D was IL-2+ cell immunization group, E group was IFN- gamma injection group, F group was IFN- y + worm cell immunization group, G group was letinous edodes polysaccharide injection group, and F group was letinous edodes polysaccharide + worm cell immune group. 2W 1 times, a total of 3 times. After the last immunization, a single blind method was used to attack the mice in each group (30 tail / rat). The observation results of the 35d caesarean section after infection were observed. The protective effect observation included counting the worm and liver eggs. The immunological observation index included: ELISA was used to detect the activity of the specific antibody in the serum of the mice before and after the immunization. [results] PCR identification showed that Schistosoma cells had no host component contamination. The protective effect showed that compared with group A, the rate of worm reduction in group B was 38.4%, and the female rate was 42.1%; the rate of worm reduction in group C, D, E, F, G and H were 13.5%, 36.1% and 32.7%, 46.2%, 18.3% and 33.7%, respectively, and the reduction rate was -2.1%, 21.1%, 38.9%, 40%, 30.5%, respectively. The LEPG reduction rates of group 40%.B, group C, group D, E, F, G, and H were 59.8%, 26.6%, 52.1%, 47.7%, 57.7%, 34% and 53.7%, and the rate of EPF reduction was 48.6%, 25.2%, 44.4%, 34.7%, 50%, 28.5% and 42.3%. immunomodulators compared with the single cell group, and the specific antibody level was not statistically significant. [Conclusion] immunization Enhancers did not significantly enhance the immune protective effect of larvae, but IFN- gamma alone had some protective effects against Schistosoma japonicum.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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