巨噬细胞内DNA-PKcs协同Aire作用调节TLRs的表达

发布时间：2018-01-03 13:29

本文关键词：巨噬细胞内DNA-PKcs协同Aire作用调节TLRs的表达　出处：《吉林大学》2012年博士论文　论文类型：学位论文

【摘要】：自身免疫调节因子（autoimmune regulator, Aire）在胸腺髓质上皮细胞（mTECs）内能够调控外周组织自身抗原（peripheral tissue self-antigen, PTA）的表达进而诱导中枢耐受。然而，，在外周淋巴组织和造血细胞内，Aire的功能还不是很明确。我们课题组前期研究表明，在稳定转染Aire的小鼠巨噬样细胞系RAW264.7（GFP-Aire/RAW）细胞内，（Toll-like receptor, TLR）1、TLR3、TLR8的表达明显升高。然而Aire影响TLR1、TLR3、TLR8表达的机制还不是很清楚。有研究报道，Aire与其协同分子DNA依赖的蛋白激酶（DNA-PK）的相互作用，是调控其转录活性的关键。因此我们推测，在巨噬细胞内，DNA-PK是否能够协同Aire作用调控TLR1、TLR3、TLR8的表达呢？为了阐明在RAW264.7细胞内Aire影响TLR1、TLR3、TLR8表达的机制，探讨DNA-PK是否能够协同Aire作用调控TLRs的表达，本课题在稳定表达Aire的小鼠巨噬细胞系RAW264.7细胞模型和瞬时转染Aire的小鼠腹腔巨噬细胞模型的基础上，从以下几方面进行了研究： 1研究DNA-PKcs与Aire的相互作用为了研究DNA-PKcs是否能够与Aire相互作用，我们采用相互免疫共沉淀和免疫荧光的方法检测RAW264.7细胞内Aire与DNA-PKcs的结合及定位情况。结果显示，在RAW细胞内，Aire能够与DNA-PKcs相互结合并与部分DNA-PKcs在细胞核中共定位。 2探讨沉默DNA-PKcs后对RAW264.7细胞内Aire调控TLRs表达的影响及其调控机制为了研究在RAW264.7细胞内DNA-PKcs对Aire调控TLRs的表达是否有影响，沉默DNA-PKcs后，我们采用RT-qPCR和FCM的方法检测TLRs的表达。结果显示，在GFP-Aire/RAW细胞内，沉默DNA-PKcs后，TLR1, TLR3和TLR8的表达明显下调。利用TLRs报告基因载体进行荧光素酶报告基因的检测。结果显示，DNA-PKcs沉默后，GFP-Aire/RAW细胞内的TLR1, TLR3, TLR8的启动子转录活性明显降低。结果表明，在RAW264.7细胞内，DNA-PKcs能够协同Aire作用调控TLR1, TLR3和TLR8的表达。 3探讨沉默DNA-PKcs后对小鼠腹腔巨噬细胞内Aire调控TLRs表达的影响为了进一步研究在小鼠腹腔巨噬细胞内DNA-PKcs对Aire调控TLRs的表达是否有影响，沉默DNA-PKcs后，我们采用RT-qPCR的方法检测TLRs的表达。结果显示，在pEGFPC1/Aire瞬时转染的细胞中，DNA-PKcs沉默后，TLR1,TLR3和TLR8的表达明显下调。结果表明，在巨噬细胞内，DNA-PKcs能够协同Aire作用调控TLR1, TLR3和TLR8的表达。本研究证明了在小鼠巨噬细胞内DNA-PKcs可协同Aire作用调控TLR1、3、8的表达，为阐明Aire调控TLR1、3、8表达的机制、进而影响对病原微生物和自身衰老病变细胞的识别提供实验依据，从而为Aire在外周表达的功能和意义提供了新的线索。
[Abstract]:Autoimmune regulator. Aire can regulate peripheral tissue self-antigen in thymic medullary epithelial cells (mTECs). However, the function of Aire in peripheral lymphoid tissue and hematopoietic cells is not clear. Toll-like receptor was detected in mouse macrophage like RAW264.7 GFP-Airerrrrrrrrr-RAWcells stably transfected with Aire. The expression of TLR3 + TLR8 was significantly increased in TLR1, but the mechanism of TLR1, TLR3, TLR8 expression in TLR1, TLR3, TLR8 was not well understood. The interaction between Aire and its co-molecular protein kinase DNA-PKK is the key to regulate its transcriptional activity, so we speculate that it is in macrophages. Can DNA-PK cooperate with Aire to regulate the expression of TLR1, TLR3 and TLR8? In order to elucidate the mechanism of Aire affecting the expression of TLR1, TLR3, TLR8 in RAW264.7 cells. To investigate whether DNA-PK can cooperate with Aire to regulate the expression of TLRs. On the basis of the mouse macrophage cell line RAW264.7 cell model which stably expressed Aire and the mouse peritoneal macrophage model transfected with Aire, we studied the following aspects:. 1. Study the interaction between DNA-PKcs and Aire In order to study whether DNA-PKcs can interact with Aire. The binding and localization of Aire to DNA-PKcs in RAW264.7 cells were detected by means of mutual immunoprecipitation and immunofluorescence. The results showed that in RAW cells. Aire can interact with DNA-PKcs and co-locate with part of DNA-PKcs in the nucleus. 2 to investigate the effect of silencing DNA-PKcs on the expression of TLRs in RAW264.7 cells by Aire and its regulatory mechanism In order to study the effect of DNA-PKcs on the expression of TLRs regulated by Aire in RAW264.7 cells, DNA-PKcs was silenced. The expression of TLRs was detected by RT-qPCR and FCM. The results showed that in GFP-Aire/RAW cells, DNA-PKcs was silenced and TLR1 was silenced. The expression of TLR3 and TLR8 was down-regulated. Luciferase reporter gene was detected by TLRs reporter gene vector. The transcriptional activity of TLR1, TLR3 and TLR8 in GFP-Aire/RAW cells was significantly decreased. The results showed that the transcriptional activity of TLR1, TLR3 and TLR8 in RAW264.7 cells was significantly decreased. DNA-PKcs can cooperate with Aire to regulate the expression of TLR1, TLR3 and TLR8. 3 to investigate the effect of silencing DNA-PKcs on the expression of TLRs in murine peritoneal macrophages regulated by Aire In order to further study the effect of DNA-PKcs on the expression of TLRs regulated by Aire in murine peritoneal macrophages, DNA-PKcs was silenced. RT-qPCR was used to detect the expression of TLRs. The results showed that the expression of TLR1 was detected after silencing of pEGFPC1/Aire transient transfection cells. The expression of TLR3 and TLR8 was down-regulated. The results showed that DNA-PKcs could regulate the expression of TLR1, TLR3 and TLR8 together with Aire in macrophages. The present study demonstrated that DNA-PKcs could regulate the expression of TLR1O3O8 in murine macrophages in coordination with Aire, and to elucidate the mechanism of Aire regulating the expression of TLR1O3O8 in murine macrophages. Furthermore, it provides experimental basis for the identification of pathogenic microorganisms and autoaging lesion cells, thus providing a new clue for the function and significance of Aire expression in peripheral area.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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