腺病毒重组CTLA4Ig和α4β7诱导的耐受性树突状细胞治疗食物过敏的研究

发布时间：2018-01-31 21:39

  本文关键词： 树突状细胞 CTLA4Ig α4β7 腺病毒 食物过敏 耐受性树突状细胞 免疫治疗　出处：《重庆医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 第一部分腺病毒重组CTLA4Ig、α4β7基因修饰树突状细胞 目的通过扩增AdCTLA4Ig和Adα4β7确定AdCTLA4Ig和Adα4β7的感染效率,构建稳定表达CTLA4Ig和α4β7基因的耐受性树突状细胞疫苗。 方法AdCTLA4Ig和Adα4β7转染293细胞扩增腺病毒,TCID50(50%组织培养感染量)法测病毒滴度。取小鼠胫骨及股骨骨髓制成细胞悬液,分离出单个核细胞,在GM-CSF及IL-4作用下培养7天,在光学显微镜下观察其形态学变化。流式细胞术(FCM)鉴定为树突状细胞后,感染AdCTLA4Ig和Adα4β7。荧光显微镜下观察目的基因表达,FCM鉴定不同阶段DC表面标志表达。 结果TCIDSO测AdCTLA4Ig和Adα4β7转染293细胞的病毒滴度分别为5.01×10~8pfu/ml、4.8×10~8pfu/ml。42%左右细胞具有CD11c表型,确定为树突状细胞。荧光显微镜下观察约80%DC表达CTLA4Ig和α4β7,当MOI为50时,感染效率最高。FCM检测结果显示经AdCTLA4Ig和Adα4β7共感染的DC表面标志CD86下降明显,而MHCⅡ无改变。 结论腺病毒重组CTLA4Ig、α4β7修饰树突状细胞可使其同时表达CTLA4Ig和α4β7两种蛋白,具有耐受性DC的表型,共刺激分子CD86表达减少,而MHCⅡ无改变。 第二部分耐受性树突状细胞预防和治疗食物过敏的实验研究 目的通过小鼠尾静脉将重组AdCTLA4Ig和Adα4β7诱导的耐受性DC回输于食物(OVA)过敏小鼠体内,观察其对食物过敏小鼠的预防、治疗效果,为临床治疗儿童食物过敏提供新的思路。 方法对象:选用无鸡蛋喂养4～6周龄的SPF级BALA/c雌鼠48只为实验对象,随机分为6组。基础致敏24小时后回输耐受性DC为预防组(P组);肠道激发4小时后回输DC为治疗组(T组);预防组和治疗组按照回输AdCTLA4IgDC和AdCTLA4Ig/Adα4β7DC分为预防1组(P1组)、预防2组(P2组)和治疗1组(T1组)、治疗2组(T2组);同时设置生理盐水为阴性对照组和OVA过敏小鼠为阳性对照组。方法:(1)观察各组症状表现;(2)HE染色及甲苯胺兰观察各组小肠组织形态学变化;(3)ELISA法检测血清中OVA特异性IgE水平;(4)免疫组化法检测小鼠小肠组织中IL-10;(5)免疫荧光法检测TGF-β表达水平;(6)免疫荧光检测小肠组织中α4β7表达。 结果T1及T2组小鼠经尾静脉注射耐受性DC对OVA介导的速发型变态反应有缓解作用,其中T1组有75%(6/8)、T2组有87.5%(7/8)过敏小鼠腹泻情况减轻;OVA特异性IgE水平明显降低;HE染色可见两组小肠绒毛中上皮细胞局灶性坏死、脱落,固有层炎症细胞浸润现象明显改善;肥大细胞聚集和脱颗粒现象改善,IL-10表达增高。P1、P2组较阳性对照组无明显改善。各组TGF-β表达无差异。免疫荧光检测T2组小鼠小肠组织中α4β7表达高于其他各组。 结论:尾静脉回输AdCTLA4Ig/Adα4β7修饰的耐受性树突状细胞证实对卵清蛋白过敏小鼠具有治疗作用,可以缓解小鼠急性发作症状,减轻肠道炎症反应,促进免疫耐受形成,但耐受性树突状细胞对食物过敏无预防作用。
[Abstract]:Part I adenovirus recombinant CTLA4 IgA, 伪 4 尾 7 gene modified dendritic cells Objective to determine the infection efficiency of AdCTLA4Ig and Ad 伪 4 尾 7 by amplification of AdCTLA4Ig and Ad 伪 4 尾 7. To construct a stable dendritic cell vaccine expressing CTLA4Ig and 伪 4 尾 7 genes. Methods AdCTLA4Ig and Ad 伪 4 尾 7 were transfected into 293 cells to amplify adenovirus. The titer of the virus was measured by TCID 50% tissue culture method. The tibia and femur bone marrow of mice were taken as cell suspensions and mononuclear cells were isolated. After cultured with GM-CSF and IL-4 for 7 days, the morphological changes were observed under optical microscope. The dendritic cells were identified as dendritic cells by flow cytometry (FCM). Infected with AdCTLA4Ig and Ad 伪 4 尾 7, the expression of target gene was observed under fluorescence microscope and the surface markers of DC were identified at different stages. Results the titers of AdCTLA4Ig and Ad 伪 4 尾 7 transfected 293 cells by TCIDSO were 5.01 脳 10 ~ (-8) pFu / ml, respectively. About 4.8 脳 10 ~ (8) pFu / ml. 42% cells had CD11c phenotype and were identified as dendritic cells. About 80% expressed CTLA4Ig and 伪 _ 4 尾 _ 7 under fluorescence microscope. When MOI was 50, the CD86 of DC co-infected with AdCTLA4Ig and Ad 伪 4 尾 7 decreased significantly. However, MHC 鈪，

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