發(fā)布時(shí)間：2018-06-03 15:21

  本文選題：樹(shù)突狀細(xì)胞 + AIRE�。� 參考：《蘇州大學(xué)》2015年碩士論文

【摘要】：目的:建立大鼠骨髓來(lái)源的樹(shù)突狀細(xì)胞體外培養(yǎng)體系,檢測(cè)腺病毒對(duì)于樹(shù)突狀細(xì)胞的感染效率,建立AIRE-DC疫苗并鑒定其性狀與生物學(xué)功能。方法:(1)取4~6周齡SD大鼠,提取大鼠骨髓細(xì)胞,經(jīng)過(guò)紅細(xì)胞裂解液去除紅細(xì)胞,貼壁細(xì)胞經(jīng)細(xì)胞因子誘導(dǎo),獲得較高純度的成熟大鼠骨髓源性樹(shù)突狀細(xì)胞(r BM-DCs),期間每日觀察樹(shù)突狀細(xì)胞(DC)形態(tài)變化;(2)構(gòu)建重組AIRE基因的腺病毒載體(Ad),通過(guò)腺病毒感染DC進(jìn)行特定基因修飾,通過(guò)流式細(xì)胞術(shù)以及免疫印跡法驗(yàn)證Ad對(duì)于懸浮生長(zhǎng)的r BM-DCs以及貼壁生長(zhǎng)的DC2.4細(xì)胞株的感染以及基因修飾效率,找出最合適的腺病毒感染復(fù)數(shù);(3)提取鼠全細(xì)胞肝癌細(xì)胞株RH-35抗原,將AIRE基因修飾的DC負(fù)載腫瘤抗原,檢測(cè)表面分子CD83、CD86、CD11c的m RNA表達(dá)水平,并通過(guò)流式細(xì)胞術(shù)檢測(cè)CD80、和MHC-II的表達(dá),研究AIRE基因?qū)τ跇?shù)突狀細(xì)胞性狀以及生物學(xué)功能影響。結(jié)果:(1)骨髓細(xì)胞懸液經(jīng)各類(lèi)處理以及細(xì)胞因子誘導(dǎo)后可獲得較高純度的DC,且隨著培養(yǎng)時(shí)間的增長(zhǎng),DC細(xì)胞逐漸增大,表現(xiàn)出明顯的樹(shù)枝樣或者細(xì)足樣分叉和突起,并從半貼壁生長(zhǎng)逐漸轉(zhuǎn)變?yōu)橘N壁生長(zhǎng);(2)腺病毒對(duì)于DC2.4細(xì)胞株以及r BM-DCs均具有較高的感染效率,且均能成功對(duì)DC進(jìn)行修飾,其中DC2.4的最適感染復(fù)數(shù)為50MOI,感染效率可達(dá)90%以上;r BM-DCs的最適感染復(fù)數(shù)為100MOI,感染效率為70%左右,更高的感染復(fù)數(shù)則會(huì)產(chǎn)生較大細(xì)胞毒性;(3)經(jīng)AIRE基因修飾過(guò)的r BM-DCs經(jīng)過(guò)腫瘤抗原負(fù)載的條件下表現(xiàn)出各類(lèi)細(xì)胞表面分子的顯著表達(dá)上升,CD80、CD83、CD11c與未處理組相比具有顯著性差異(p0.05),CD86和MHC-II類(lèi)分子與未處理組相比差異非常顯著(p0.01),同時(shí)轉(zhuǎn)染空載體的r BM-DCs表面分子與為處理組相比無(wú)顯著性差異。而DC2.4在感染前后表面分子表達(dá)均較低且無(wú)顯著性差異。結(jié)論:1.腺病毒對(duì)于原代DC以及DC細(xì)胞株均具較高的感染效率,但對(duì)于細(xì)胞株的感染效率更高;2.腺病毒對(duì)r BM-DCs的最適感染復(fù)數(shù)較DC2.4高,懸浮細(xì)胞相對(duì)于貼壁細(xì)胞較難感染;3.AIRE可以增高負(fù)載抗原后的r BM-DCs表面分子CD80、CD83、CD86、CD11c和MHC-II類(lèi)表達(dá),其中CD86和MHC-II類(lèi)分子表達(dá)顯著增高。4.DC2.4細(xì)胞株缺乏DC細(xì)胞應(yīng)該表達(dá)的各類(lèi)表面分子,且腫瘤全細(xì)胞抗原刺激后表達(dá)亦沒(méi)有顯著變化,表明其經(jīng)過(guò)正常細(xì)胞永生化處理,且長(zhǎng)期傳代后存在DC生物學(xué)功能的喪失。
[Abstract]:Aim: to establish a culture system of rat bone marrow-derived dendritic cells in vitro, to detect the infection efficiency of adenovirus on dendritic cells, to establish AIRE-DC vaccine and to identify its characters and biological functions. Methods Sprague-Dawley rats aged 4 ~ 6 weeks were taken from SD rats. Bone marrow cells were extracted from SD rats. Erythrocytes were removed by erythrocyte lysate, and adherent cells were induced by cytokines. High purity mature rat bone marrow-derived dendritic cells were obtained. During the period, morphological changes of dendritic cells were observed daily. The adenovirus vector of recombinant AIRE gene was constructed and modified by adenovirus infected DC. The infection and gene modification efficiency of Ad against suspended r BM-DCs and adherent DC2.4 cell lines were verified by flow cytometry and Western blotting. To find out the most suitable adenovirus infection, RH-35 antigen was extracted from the whole cell hepatoma cell line, the tumor antigen was loaded with DC modified by AIRE gene, the expression level of m RNA of CD83 + CD86 + CD11c was detected, and the expression of CD80, and MHC-II were detected by flow cytometry. To study the effects of AIRE gene on dendritic cell traits and biological functions. Results (1) Bone marrow cell suspensions were treated with various treatments and induced by cytokines to obtain high purity DC.The DC cells increased gradually with the increase of culture time, showing obvious branching and protruding of dendritic or thin foot-like cells. And the adenovirus transformed from semi-adherent growth to adherent growth 2) adenovirus had higher infection efficiency to DC2.4 cell line and r BM-DCs, and could modify DC successfully. The optimal infection number of DC2.4 is 50 moi, the infection efficiency is more than 90%, the optimal number of infection is 100MOI, and the infection efficiency is about 70%. The higher the complex number of infection is, the larger cytotoxicity is produced. The r BM-DCs modified by AIRE gene shows a significant increase in the expression of various cell surface molecules under the condition of tumor antigen loading. CD80, CD83 and CD11c are significantly higher than those of the untreated group. There were significant differences in p0.05, CD86 and MHC-II molecules between the untreated group and the untreated group. There was no significant difference in the surface molecules of r BM-DCs transfected with the empty vector as compared with the treated group. However, there was no significant difference in the expression of DC2.4 on the surface before and after infection. Conclusion 1. Adenovirus had higher infection efficiency to primary DC and DC cell lines, but higher infection efficiency to cell lines. The optimal number of adenovirus infection to r BM-DCs is higher than that of DC2.4. Compared with adherent cells, immune can increase the expression of CD80, CD83, CD86, CD11c and MHC-II on the surface of r BM-DCs. The expression of CD86 and MHC-II class molecules increased significantly. 4. DC2.4 cell line lacked all kinds of surface molecules that should be expressed by DC cells, and the expression of tumor whole cell antigen was not changed after stimulation, indicating that the cells were treated with immortalization of normal cells. The biological function of DC was lost after long passage.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R730.51

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