Th17细胞在移植物抗宿主病中的作用和基因工程Th17细胞的制备及鉴定

发布时间：2018-07-07 17:22

本文选题：Th17 + 细胞　；参考：《南京医科大学》2012年博士论文

【摘要】：本论文分为两个部分。第一部分主要探讨Th17细胞及白细胞介素-17（IL-17）与小鼠异基因造血干细胞移植后移植物抗宿主病（GVHD）之间的关系；第二部分则探索了一种基于基因工程技术制备和分选Th17细胞的新方法。 Th17细胞是近年提出的一个辅助性T细胞新亚群，主要通过分泌IL-17等细胞因子发挥功能，与抗感染免疫和自身免疫病发生密切相关。然而，Th17细胞与GVHD的关系至今仍有很大争论。在第一部分研究中，我们首先通过输注不同的脾细胞量建立了重度和轻-中度GVHD小鼠模型，发现外周血Th17细胞的比率和IL-17水平与GVHD严重程度成负相关关系。我们进一步细化了allo-HSCT后CD4+T细胞亚群失衡与不同靶脏器损伤的相关性，发现Th1细胞与肝脏损伤相关，Th17细胞与肺损伤相关，Th1/Th17细胞比率与小肠损伤相关。为了进一步验证Th17细胞与GVHD相关肺损伤的关系，我们使用了肺特异性高表达IL-17的转基因小鼠作为受鼠建立GVHD模型，，不仅证实IL-17是GVHD相关肺损伤的促进因素，而且发现IL-17系通过促进肺组织表达趋化因子增加炎症细胞招募和促进Th1细胞分化加重GVHD相关肺损伤，初步阐明了其作用机制。 Th17细胞由于含量很低且无膜表面特异性标志或标记组合，又由于其发育调控的复杂性导致体外诱导产率不高而难以制备和纯化。在第二部分研究中，我们尝试将过表达Th17细胞发育的主要正调控因子RORγt与TGF-β+IL-6细胞因子体外处理联合起来，使naive CD4+T细胞获得了更高的Th17细胞得率。此外，我们在载体上负载了IL-17启动子驱动的截短型人神经生长因子受体（ΔNGFR）报告基因，通过分选ΔNGFR+细胞纯化了制备的基因工程Th17细胞。表型和功能鉴定表明，过表达RORγt联合TGF-β+IL-6细胞因子体外处理制备的Th17细胞较单纯过表达RORγt制备的Th17细胞更接近天然的Th17细胞，可为Th17细胞功能的研究提供有效的工具。
[Abstract]:This paper is divided into two parts. In the first part, the relationship between Th17 cells, interleukin-17 (IL-17) and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation was studied. In the second part, a new method for preparing and sorting Th17 cells based on genetic engineering is explored. Th17 cells are a new helper T cell subgroup proposed in recent years. Th17 cells play a role by secreting cytokines such as IL-17. It is closely related to the occurrence of anti-infection immunity and autoimmune disease. However, the relationship between Th17 cells and GVHD is still controversial. In the first part of the study, we first established the severe and mild-moderate GVHD model by injecting different amount of spleen cells. We found that the ratio of Th17 cells and IL-17 level in peripheral blood were negatively correlated with the severity of GVHD. We further refined the relationship between the imbalance of CD4 T cell subsets after allo-HSCT and the injury of different target organs. It was found that the ratio of Th1 cells and liver injury related to Th17 cells and lung injury correlated with the ratio of Th1 / Th17 cells and intestinal injury. In order to further verify the relationship between Th17 cells and GVHD-associated lung injury, we used transgenic mice with lung specific high expression of IL-17 as a GVHD model to establish GVHD model, which not only confirmed that IL-17 was the promoting factor of GVHD-associated lung injury. It was also found that IL-17 increased the recruitment of inflammatory cells and enhanced the differentiation of Th1 cells by promoting the expression of chemokines in lung tissue. Th17 cells are difficult to be prepared and purified due to their low content and lack of membrane surface specific markers or marker combinations, and because of the complexity of their developmental regulation, resulting in a low induced yield in vitro. In the second part of the study, we try to combine ROR 纬 t, the main positive regulatory factor of over-expression Th17 cell development, with TGF- 尾 IL-6 cytokines in vitro, so that naive CD4 T cells can obtain higher Th17 cell yield. In addition, we loaded truncated human nerve growth factor receptor (螖 NGFR) reporter gene driven by IL-17 promoter on the vector and purified the engineered Th17 cells by sorting 螖 NGFR cells. Phenotypic and functional identification showed that Th17 cells prepared by overexpression of ROR 纬 t combined with TGF- 尾 IL-6 cytokines were closer to natural Th17 cells than those prepared by over-expressing ROR 纬 t in vitro, which could provide an effective tool for the study of Th17 cell function.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.4

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