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

【摘要】： 第一部分腺病毒重組CTLA4Ig、α4β7的擴增及轉染樹突狀細胞 目的通過擴增AdCTLA4Ig和Adα4β7確定AdCTLA4Ig和Adα4β7的感染效率,構建穩(wěn)定表達CTLA4Ig和α4β7基因的耐受性樹突狀細胞疫苗。 方法AdCTLA4Ig和Adα4β7轉染293細胞擴增腺病毒,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胞懸液,分離出單個核細胞,在GM-CSF及IL-4作用下培養(yǎng)7天,在光學顯微鏡下觀察其形態(tài)學變化。流式細胞術(FCM)鑒定為樹突狀細胞后,感染AdCTLA4Ig和Adα4β7。熒光顯微鏡下觀察目的基因表達,FCM鑒定不同階段DC表面標志表達。 結果TCID50測AdCTLA4Ig和Adα4β7轉染293細胞的病毒滴度分別為5.01×108pfu/ml、4.8×108pfu/ml。42%左右細胞具有CD11c表型,確定為樹突狀細胞。熒光顯微鏡下觀察約80%DC表達CTLA4Ig和α4β7,當MOI為50時,感染效率最高。FCM檢測結果顯示經(jīng)AdCTLA4Ig和Adα4β7共感染的DC表面標志CD86下降明顯,而MHCⅡ無改變。 結論腺病毒重組CTLA4Ig、α4β7修飾樹突狀細胞可使其同時表達CTLA4Ig和α4β7兩種蛋白,具有耐受性DC的表型,共刺激分子CD86表達減少,而MHCⅡ無改變。 第二部分耐受性樹突狀細胞預防和治療食物過敏的實驗研究 目的通過小鼠尾靜脈將重組AdCTLA4Ig和Adα4β7誘導的耐受性DC回輸于食物(OVA)過敏小鼠體內,觀察其對食物過敏小鼠的預防、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只為實驗對象,隨機分為6組�；A致敏24小時后回輸耐受性DC為預防組(P組);腸道激發(fā)4小時后回輸DC為治療組(T組);預防組和治療組按照回輸AdCTLA4IgDC和AdCTLA4Ig/Adα4β7DC分為預防1組(P1組)、預防2組(P2組)和治療1組(T1組)、治療2組(T2組);同時設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表達水平;(6)免疫熒光檢測小腸組織中α4β7表達。 結果T1及T2組小鼠經(jīng)尾靜脈注射耐受性DC對OVA介導的速發(fā)型變態(tài)反應有緩解作用,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胞局灶性壞死、脫落,固有層炎癥細胞浸潤現(xiàn)象明顯改善;肥大細胞聚集和脫顆粒現(xiàn)象改善,IL-10表達增高。P1、P2組較陽性對照組無明顯改善。各組TGF-β表達無差異。免疫熒光檢測T2組小鼠小腸組織中α4β7表達高于其他各組。 結論:尾靜脈回輸AdCTLA4Ig/Adα4β7修飾的耐受性樹突狀細胞證實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,促進免疫耐受形成,但耐受性樹突狀細胞對食物過敏無預防作用。
[Abstract]:Part I Amplification of adenovirus Recombinant CTLA4 IgA, 偽 4 尾 7 and transfection of dendritic cells. Objective to determine the infection efficiency of AdCTLA4Ig and Ad 偽 4 尾 7 by amplifying AdCTLA4Ig and Ad 偽 4 尾 7, and to construct a resistant dendritic cell vaccine expressing CTLA4Ig and 偽 4 尾 7 genes stably. Methods the titer of AdCTLA4Ig and Ad 偽 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 偽 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 偽 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 偽 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 偽 4 尾 7 decreased significantly, but MHC 鈪，

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