本文關(guān)鍵詞：巨噬細(xì)胞內(nèi)DNA-PKcs協(xié)同Aire作用調(diào)節(jié)TLRs的表達(dá)　出處：《吉林大學(xué)》2012年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： Aire 巨噬細(xì)胞 Toll樣受體 協(xié)同分子 DNA-PKcs

【摘要】：自身免疫調(diào)節(jié)因子（autoimmune regulator, Aire）在胸腺髓質(zhì)上皮細(xì)胞（mTECs）內(nèi)能夠調(diào)控外周組織自身抗原（peripheral tissue self-antigen, PTA）的表達(dá)進(jìn)而誘導(dǎo)中樞耐受。然而，，在外周淋巴組織和造血細(xì)胞內(nèi)，Aire的功能還不是很明確。我們課題組前期研究表明，在穩(wěn)定轉(zhuǎn)染Aire的小鼠巨噬樣細(xì)胞系RAW264.7（GFP-Aire/RAW）細(xì)胞內(nèi)，（Toll-like receptor, TLR）1、TLR3、TLR8的表達(dá)明顯升高。然而Aire影響TLR1、TLR3、TLR8表達(dá)的機(jī)制還不是很清楚。有研究報(bào)道，Aire與其協(xié)同分子DNA依賴(lài)的蛋白激酶（DNA-PK）的相互作用，是調(diào)控其轉(zhuǎn)錄活性的關(guān)鍵。因此我們推測(cè)，在巨噬細(xì)胞內(nèi)，DNA-PK是否能夠協(xié)同Aire作用調(diào)控TLR1、TLR3、TLR8的表達(dá)呢？ 為了闡明在RAW264.7細(xì)胞內(nèi)Aire影響TLR1、TLR3、TLR8表達(dá)的機(jī)制，探討DNA-PK是否能夠協(xié)同Aire作用調(diào)控TLRs的表達(dá)，本課題在穩(wěn)定表達(dá)Aire的小鼠巨噬細(xì)胞系RAW264.7細(xì)胞模型和瞬時(shí)轉(zhuǎn)染Aire的小鼠腹腔巨噬細(xì)胞模型的基礎(chǔ)上，從以下幾方面進(jìn)行了研究： 1研究DNA-PKcs與Aire的相互作用 為了研究DNA-PKcs是否能夠與Aire相互作用，我們采用相互免疫共沉淀和免疫熒光的方法檢測(cè)RAW264.7細(xì)胞內(nèi)Aire與DNA-PKcs的結(jié)合及定位情況。結(jié)果顯示，在RAW細(xì)胞內(nèi)，Aire能夠與DNA-PKcs相互結(jié)合并與部分DNA-PKcs在細(xì)胞核中共定位。 2探討沉默DNA-PKcs后對(duì)RAW264.7細(xì)胞內(nèi)Aire調(diào)控TLRs表達(dá)的影響及其調(diào)控機(jī)制 為了研究在RAW264.7細(xì)胞內(nèi)DNA-PKcs對(duì)Aire調(diào)控TLRs的表達(dá)是否有影響，沉默DNA-PKcs后，我們采用RT-qPCR和FCM的方法檢測(cè)TLRs的表達(dá)。結(jié)果顯示，在GFP-Aire/RAW細(xì)胞內(nèi)，沉默DNA-PKcs后，TLR1, TLR3和TLR8的表達(dá)明顯下調(diào)。利用TLRs報(bào)告基因載體進(jìn)行熒光素酶報(bào)告基因的檢測(cè)。結(jié)果顯示，DNA-PKcs沉默后，GFP-Aire/RAW細(xì)胞內(nèi)的TLR1, TLR3, TLR8的啟動(dòng)子轉(zhuǎn)錄活性明顯降低。結(jié)果表明，在RAW264.7細(xì)胞內(nèi)，DNA-PKcs能夠協(xié)同Aire作用調(diào)控TLR1, TLR3和TLR8的表達(dá)。 3探討沉默DNA-PKcs后對(duì)小鼠腹腔巨噬細(xì)胞內(nèi)Aire調(diào)控TLRs表達(dá)的影響 為了進(jìn)一步研究在小鼠腹腔巨噬細(xì)胞內(nèi)DNA-PKcs對(duì)Aire調(diào)控TLRs的表達(dá)是否有影響，沉默DNA-PKcs后，我們采用RT-qPCR的方法檢測(cè)TLRs的表達(dá)。結(jié)果顯示，在pEGFPC1/Aire瞬時(shí)轉(zhuǎn)染的細(xì)胞中，DNA-PKcs沉默后，TLR1,TLR3和TLR8的表達(dá)明顯下調(diào)。結(jié)果表明，在巨噬細(xì)胞內(nèi)，DNA-PKcs能夠協(xié)同Aire作用調(diào)控TLR1, TLR3和TLR8的表達(dá)。 本研究證明了在小鼠巨噬細(xì)胞內(nèi)DNA-PKcs可協(xié)同Aire作用調(diào)控TLR1、3、8的表達(dá)，為闡明Aire調(diào)控TLR1、3、8表達(dá)的機(jī)制、進(jìn)而影響對(duì)病原微生物和自身衰老病變細(xì)胞的識(shí)別提供實(shí)驗(yàn)依據(jù)，從而為Aire在外周表達(dá)的功能和意義提供了新的線(xiàn)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 朱武飛;自身免疫調(diào)節(jié)因子對(duì)巨噬細(xì)胞TLRs表達(dá)及其活化類(lèi)型的影響[D];吉林大學(xué);2011年

本文編號(hào)：1374097