本文關(guān)鍵詞： 樹突狀細(xì)胞 CTLA4Ig α4β7 腺病毒 食物過敏 耐受性樹突狀細(xì)胞 免疫治療　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 第一部分腺病毒重組CTLA4Ig、α4β7基因修飾樹突狀細(xì)胞 目的通過擴(kuò)增AdCTLA4Ig和Adα4β7確定AdCTLA4Ig和Adα4β7的感染效率,構(gòu)建穩(wěn)定表達(dá)CTLA4Ig和α4β7基因的耐受性樹突狀細(xì)胞疫苗。 方法AdCTLA4Ig和Adα4β7轉(zhuǎn)染293細(xì)胞擴(kuò)增腺病毒,TCID50(50%組織培養(yǎng)感染量)法測病毒滴度。取小鼠脛骨及股骨骨髓制成細(xì)胞懸液,分離出單個(gè)核細(xì)胞,在GM-CSF及IL-4作用下培養(yǎng)7天,在光學(xué)顯微鏡下觀察其形態(tài)學(xué)變化。流式細(xì)胞術(shù)(FCM)鑒定為樹突狀細(xì)胞后,感染AdCTLA4Ig和Adα4β7。熒光顯微鏡下觀察目的基因表達(dá),FCM鑒定不同階段DC表面標(biāo)志表達(dá)。 結(jié)果TCIDSO測AdCTLA4Ig和Adα4β7轉(zhuǎn)染293細(xì)胞的病毒滴度分別為5.01×10~8pfu/ml、4.8×10~8pfu/ml。42%左右細(xì)胞具有CD11c表型,確定為樹突狀細(xì)胞。熒光顯微鏡下觀察約80%DC表達(dá)CTLA4Ig和α4β7,當(dāng)MOI為50時(shí),感染效率最高。FCM檢測結(jié)果顯示經(jīng)AdCTLA4Ig和Adα4β7共感染的DC表面標(biāo)志CD86下降明顯,而MHCⅡ無改變。 結(jié)論腺病毒重組CTLA4Ig、α4β7修飾樹突狀細(xì)胞可使其同時(shí)表達(dá)CTLA4Ig和α4β7兩種蛋白,具有耐受性DC的表型,共刺激分子CD86表達(dá)減少,而MHCⅡ無改變。 第二部分耐受性樹突狀細(xì)胞預(yù)防和治療食物過敏的實(shí)驗(yàn)研究 目的通過小鼠尾靜脈將重組AdCTLA4Ig和Adα4β7誘導(dǎo)的耐受性DC回輸于食物(OVA)過敏小鼠體內(nèi),觀察其對食物過敏小鼠的預(yù)防、治療效果,為臨床治療兒童食物過敏提供新的思路。 方法對象:選用無雞蛋喂養(yǎng)4～6周齡的SPF級BALA/c雌鼠48只為實(shí)驗(yàn)對象,隨機(jī)分為6組。基礎(chǔ)致敏24小時(shí)后回輸耐受性DC為預(yù)防組(P組);腸道激發(fā)4小時(shí)后回輸DC為治療組(T組);預(yù)防組和治療組按照回輸AdCTLA4IgDC和AdCTLA4Ig/Adα4β7DC分為預(yù)防1組(P1組)、預(yù)防2組(P2組)和治療1組(T1組)、治療2組(T2組);同時(shí)設(shè)置生理鹽水為陰性對照組和OVA過敏小鼠為陽性對照組。方法:(1)觀察各組癥狀表現(xiàn);(2)HE染色及甲苯胺蘭觀察各組小腸組織形態(tài)學(xué)變化;(3)ELISA法檢測血清中OVA特異性IgE水平;(4)免疫組化法檢測小鼠小腸組織中IL-10;(5)免疫熒光法檢測TGF-β表達(dá)水平;(6)免疫熒光檢測小腸組織中α4β7表達(dá)。 結(jié)果T1及T2組小鼠經(jīng)尾靜脈注射耐受性DC對OVA介導(dǎo)的速發(fā)型變態(tài)反應(yīng)有緩解作用,其中T1組有75%(6/8)、T2組有87.5%(7/8)過敏小鼠腹瀉情況減輕;OVA特異性IgE水平明顯降低;HE染色可見兩組小腸絨毛中上皮細(xì)胞局灶性壞死、脫落,固有層炎癥細(xì)胞浸潤現(xiàn)象明顯改善;肥大細(xì)胞聚集和脫顆�，F(xiàn)象改善,IL-10表達(dá)增高。P1、P2組較陽性對照組無明顯改善。各組TGF-β表達(dá)無差異。免疫熒光檢測T2組小鼠小腸組織中α4β7表達(dá)高于其他各組。 結(jié)論:尾靜脈回輸AdCTLA4Ig/Adα4β7修飾的耐受性樹突狀細(xì)胞證實(shí)對卵清蛋白過敏小鼠具有治療作用,可以緩解小鼠急性發(fā)作癥狀,減輕腸道炎癥反應(yīng),促進(jìn)免疫耐受形成,但耐受性樹突狀細(xì)胞對食物過敏無預(yù)防作用。
[Abstract]:Part I adenovirus recombinant CTLA4 IgA, 偽 4 尾 7 gene modified dendritic cells Objective to determine the infection efficiency of AdCTLA4Ig and Ad 偽 4 尾 7 by amplification of AdCTLA4Ig and Ad 偽 4 尾 7. To construct a stable dendritic cell vaccine expressing CTLA4Ig and 偽 4 尾 7 genes. Methods AdCTLA4Ig and Ad 偽 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 偽 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 偽 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 偽 _ 4 尾 _ 7 under fluorescence microscope. When MOI was 50, the CD86 of DC co-infected with AdCTLA4Ig and Ad 偽 4 尾 7 decreased significantly. However, MHC 鈪，

本文編號(hào)：1480060