重组腺病毒介导的IDO高表达DC对同种反应T细胞亚群分化的影响

发布时间：2018-02-04 18:36

本文关键词： IDO 骨髓来源树突状细胞同种皮肤移植移植排斥反应　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：目的:以重组腺病毒为IDO表达载体(Ad-IDO),感染供者骨髓来源的DC,将其过继输注给受者,观察高表达IDO的DC输注对受者皮肤移植后体内T细胞亚群分化的影响。同时在体外进行混合淋巴细胞培养,检测同种反应T细胞亚群的分化特征,为阐明IDO抑制同种反应T细胞分化而保护移植物提供实验依据。方法:以293T细胞为包装细胞,扩增重组的Ad-IDO(E1、E3区缺失;外源基因插入E1区;以CMV为启动子,以SV40polyA为加尾信号)。将扩增的Ad-IDO感染供者骨髓来源的DC,与异基因受者小鼠来源的脾脏na?ve T细胞进行共培养,用流式细胞术检测培养体系中同种反应T细胞亚群(Th1、Th17)的比例;将Ad-IDO转染的供者骨髓来源的DC输注异基因受者小鼠,对受者进行皮肤移植,并在移植后取受者的脾脏、淋巴结细胞进行流式检测,以观察其T细胞亚群的比例变化。结果:一、用293T细胞扩增重组腺病毒,并对其进行鉴定。在293T细胞的连续传代培养过程中,Ad-IDO以及Ad对照稳定转染,细胞表现出明显的病变效应;PCR表明,重组腺病毒在多次复制后仍能稳定整合IDO基因;免疫印迹结果表明,不同代数的Ad-IDO感染293T细胞,均能使其稳定表达IDO蛋白。二、高表达IDO的供者骨髓来源DC对受者T细胞亚群分化的影响 1、BMDC的培养,腺病毒感染及IDO的表达诱导培养BALB/c小鼠来源的BMDC,并用Ad-IDO及对照组腺病毒进行感染。经RT-PCR鉴定,感染了Ad-IDO的BMDC有IDO的mRNA表达;经Western Blot鉴定,感染后的BMDC可有效表达IDO蛋白。 2、IDO及其代谢产物L-Kyn抑制同种反应Th17与Th1分化将感染了Ad-IDO的BMDC与对照组BMDC(BALB/c小鼠来源)分别与C57BL/6脾脏来源的T细胞进行混合淋巴细胞培养,并通过流式细胞术检测。与BMDC及BMDC(Ad-control)组相比,BMDC(Ad-IDO)组的Th17及Th1明显降低;同时设立了加入L-Kyn(IDO降解色氨酸的代谢产物)的组别,其Th17和Th1的比例也明显降低。 3、体内输注供者来源IDO高表达DC显著抑制同种反应性Th1而对Th17无明显影响将BALB/c小鼠来源的BMDC过继输注给C57BL/6小鼠,并进行背部的皮肤移植,于移植后不同时间点取受者的脾脏、引流淋巴结,检测其T细胞亚型(Th17、Th1)的比例。与对照组相比,过继输注了感染Ad-IDO的BMDC组的小鼠其脾脏Th1比例明显降低,而Th17处于极低水平,尚不能评价其在各组间的差异。受者淋巴结的T细胞亚群在不同组别间的差距并不明显。结论:以重组腺病毒为载体感染供者骨髓来源的树突状细胞,使其高表达IDO,可抑制不同品系来源的Th1和Th17的分化。之前的研究已经证实,将其过继输注给受者并进行皮肤移植,可以延长皮肤移植物的存活时间;在本研究中,通过检测受者同种反应性T细胞亚群的变化来进一步探讨了其机制。本研究成果提示,使供者骨髓来源树突状细胞高表达IDO,并过继输注可进一步抑制同种移植物排斥反应,具有潜在的临床应用价值。
[Abstract]:Objective: IDO expression vector for recombinant adenovirus (Ad-IDO) infection, donor bone marrow derived DC, the adoptive infusion to patients, to observe the high expression of IDO DC infusion on body skin transplantation T cell subsets in vitro. At the same time mixed lymphocyte culture in vitro, differentiation characteristics T lymphocyte subsets, to elucidate the IDO inhibitory response of T cells and protect the differentiation of allogeneic graft to provide experimental basis.
Methods: using 293T cells as packaging cells, amplification of recombinant Ad-IDO (E1, E3 deletion; gene E1; CMV promoter, SV40polyA signal). The amplified Ad-IDO infected donor bone marrow derived DC and allogeneic recipients from mouse spleen Na ve? T cells were co cultured with T cell culture system in detection of allogeneic subsets by flow cytometry (Th1, Th17) the proportion of Ad-IDO; transfection of donor bone marrow infusion of allogeneic DC recipients mice skin transplantation on recipients and recipients after transplantation in the spleen. Lymph node cells were detected by flow cytometry, to observe the subsets of T cell percentage change.
Results: 1. The recombinant adenovirus was amplified with 293T cells and identified.
In the continuous passage of 293T cells cultured in Ad-IDO and Ad control transfected cells showed obvious cytopathic effect; PCR showed that the recombinant adenovirus can still stable integration of IDO gene in multiple copies; Western blot results showed that the different passages of Ad-IDO infected 293T cells were able to make the stable expression of IDO protein.
Two, the effect of donor bone marrow DC on the differentiation of T cell subsets of the recipient with high expression of IDO
1, BMDC culture, adenovirus infection and expression of IDO
The BMDC from BALB/c mice was induced and cultured, and infected by adenovirus and Ad-IDO. After RT-PCR identification, BMDC infected with Ad-IDO had IDO mRNA expression. After Western Blot identification, the infected BMDC could effectively express the Western protein.
2, IDO and its metabolite L-Kyn inhibit the differentiation of homologous Th17 and Th1
The infected Ad-IDO BMDC and control group BMDC (BALB/c mouse source) C57BL/6 respectively with spleen derived T cells in mixed lymphocyte culture, and were analyzed by flow cytometry. BMDC and BMDC (Ad-control) group, BMDC (Ad-IDO) Th17 and Th1 were significantly reduced; while the establishment of the accession to the L-Kyn (the degradation of IDO metabolites of tryptophan) group, the Th17 and Th1 ratio were significantly decreased.
3, high expression of DC in the donor source IDO significantly inhibited the homoreactivity of Th1 but had no significant effect on Th17
The BALB/c mouse derived BMDC adoptive transfer to C57BL/6 mice, and the skin on the back of the transplant, to take the different time points after transplantation of spleen, lymph nodes, the detection of T cell subsets (Th17, Th1) ratio. Compared with the control group, the adoptive transfer of Ad-IDO infection in BMDC group the mice spleen Th1 ratio decreased significantly, while Th17 at a very low level, not in the evaluation of the differences among the groups. The lymph node of T cell subsets in different groups of the gap is not obvious.
Conclusion: the recombinant adenovirus infected donor bone marrow derived dendritic cells, the high expression of IDO can inhibit the differentiation of different strains from Th1 and Th17. Previous studies have confirmed that the adoptive transfer to recipients and skin transplantation can prolong skin graft survival; in this study, by detecting the recipients of allogeneic T cells subsets to further explore its mechanism. The results of this study suggest that donor bone marrow derived dendritic cells with high expression of IDO, and the adoptive infusion can further inhibit allograft rejection and potential clinical value.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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