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体内研究

发布时间：2018-02-08 09:00

  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的扩增及转染树突状细胞 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TCID50测AdCTLA4Ig和Adα4β7转染293细胞的病毒滴度分别为5.01×108pfu/ml、4.8×108pfu/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 Amplification of adenovirus Recombinant CTLA4 IgA, 伪 4 尾 7 and transfection of dendritic cells. Objective to determine the infection efficiency of AdCTLA4Ig and Ad 伪 4 尾 7 by amplifying AdCTLA4Ig and Ad 伪 4 尾 7, and to construct a resistant dendritic cell vaccine expressing CTLA4Ig and 伪 4 尾 7 genes stably. Methods the titer of AdCTLA4Ig and Ad 伪 4 尾 7 transfected 293 cells was measured by TCID50 50% tissue culture method. The tibia and femur bone marrow of mice were extracted into cell suspensions. Mononuclear cells were isolated and cultured under the action of GM-CSF and IL-4 for 7 days. The morphological changes were observed under optical microscope. The dendritic cells were identified by flow cytometry (FCM) and 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 by FCM at different stages. Results the viral titers of AdCTLA4Ig and Ad 伪 4 尾 7 transfected 293 cells were 5.01 脳 10 8 pFuP / ml 路42%, respectively, and the cells were identified as dendritic cells. About 80 cells expressed CTLA4Ig and 伪 4 尾 7 were observed under fluorescence microscope, and when MOI was 50, the cells had CD11c phenotype. The results showed that the CD86 of DC co-infected with AdCTLA4Ig and Ad 伪 4 尾 7 decreased significantly, but MHC 鈪，

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