腺病毒介导的自身免疫调节因子（AIRE）基因修饰对树突状细胞性状及功能影响的研究

发布时间：2018-06-03 15:21

本文选题：树突状细胞 + AIRE　；参考：《苏州大学》2015年硕士论文

【摘要】：目的:建立大鼠骨髓来源的树突状细胞体外培养体系,检测腺病毒对于树突状细胞的感染效率,建立AIRE-DC疫苗并鉴定其性状与生物学功能。方法:(1)取4~6周龄SD大鼠,提取大鼠骨髓细胞,经过红细胞裂解液去除红细胞,贴壁细胞经细胞因子诱导,获得较高纯度的成熟大鼠骨髓源性树突状细胞(r BM-DCs),期间每日观察树突状细胞(DC)形态变化;(2)构建重组AIRE基因的腺病毒载体(Ad),通过腺病毒感染DC进行特定基因修饰,通过流式细胞术以及免疫印迹法验证Ad对于悬浮生长的r BM-DCs以及贴壁生长的DC2.4细胞株的感染以及基因修饰效率,找出最合适的腺病毒感染复数;(3)提取鼠全细胞肝癌细胞株RH-35抗原,将AIRE基因修饰的DC负载肿瘤抗原,检测表面分子CD83、CD86、CD11c的m RNA表达水平,并通过流式细胞术检测CD80、和MHC-II的表达,研究AIRE基因对于树突状细胞性状以及生物学功能影响。结果:(1)骨髓细胞悬液经各类处理以及细胞因子诱导后可获得较高纯度的DC,且随着培养时间的增长,DC细胞逐渐增大,表现出明显的树枝样或者细足样分叉和突起,并从半贴壁生长逐渐转变为贴壁生长;(2)腺病毒对于DC2.4细胞株以及r BM-DCs均具有较高的感染效率,且均能成功对DC进行修饰,其中DC2.4的最适感染复数为50MOI,感染效率可达90%以上;r BM-DCs的最适感染复数为100MOI,感染效率为70%左右,更高的感染复数则会产生较大细胞毒性;(3)经AIRE基因修饰过的r BM-DCs经过肿瘤抗原负载的条件下表现出各类细胞表面分子的显著表达上升,CD80、CD83、CD11c与未处理组相比具有显著性差异(p0.05),CD86和MHC-II类分子与未处理组相比差异非常显著(p0.01),同时转染空载体的r BM-DCs表面分子与为处理组相比无显著性差异。而DC2.4在感染前后表面分子表达均较低且无显著性差异。结论:1.腺病毒对于原代DC以及DC细胞株均具较高的感染效率,但对于细胞株的感染效率更高;2.腺病毒对r BM-DCs的最适感染复数较DC2.4高,悬浮细胞相对于贴壁细胞较难感染;3.AIRE可以增高负载抗原后的r BM-DCs表面分子CD80、CD83、CD86、CD11c和MHC-II类表达,其中CD86和MHC-II类分子表达显著增高。4.DC2.4细胞株缺乏DC细胞应该表达的各类表面分子,且肿瘤全细胞抗原刺激后表达亦没有显著变化,表明其经过正常细胞永生化处理,且长期传代后存在DC生物学功能的丧失。
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.51

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