本文關鍵詞：巨噬細胞內DNA-PKcs協(xié)同Aire作用調節(jié)TLRs的表達　出處：《吉林大學》2012年博士論文　論文類型：學位論文

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

【摘要】：自身免疫調節(jié)因子（autoimmune regulator, Aire）在胸腺髓質上皮細胞（mTECs）內能夠調控外周組織自身抗原（peripheral tissue self-antigen, PTA）的表達進而誘導中樞耐受。然而，，在外周淋巴組織和造血細胞內，Aire的功能還不是很明確。我們課題組前期研究表明，在穩(wěn)定轉染Aire的小鼠巨噬樣細胞系RAW264.7（GFP-Aire/RAW）細胞內，（Toll-like receptor, TLR）1、TLR3、TLR8的表達明顯升高。然而Aire影響TLR1、TLR3、TLR8表達的機制還不是很清楚。有研究報道，Aire與其協(xié)同分子DNA依賴的蛋白激酶（DNA-PK）的相互作用，是調控其轉錄活性的關鍵。因此我們推測，在巨噬細胞內，DNA-PK是否能夠協(xié)同Aire作用調控TLR1、TLR3、TLR8的表達呢？ 為了闡明在RAW264.7細胞內Aire影響TLR1、TLR3、TLR8表達的機制，探討DNA-PK是否能夠協(xié)同Aire作用調控TLRs的表達，本課題在穩(wěn)定表達Aire的小鼠巨噬細胞系RAW264.7細胞模型和瞬時轉染Aire的小鼠腹腔巨噬細胞模型的基礎上，從以下幾方面進行了研究： 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RAW264.7細胞內Aire與DNA-PKcs的結合及定位情況。結果顯示，在RAW細胞內，Aire能夠與DNA-PKcs相互結合并與部分DNA-PKcs在細胞核中共定位。 2探討沉默DNA-PKcs后對RAW264.7細胞內Aire調控TLRs表達的影響及其調控機制 為了研究在RAW264.7細胞內DNA-PKcs對Aire調控TLRs的表達是否有影響，沉默DNA-PKcs后，我們采用RT-qPCR和FCM的方法檢測TLRs的表達。結果顯示，在GFP-Aire/RAW細胞內，沉默DNA-PKcs后，TLR1, TLR3和TLR8的表達明顯下調。利用TLRs報告基因載體進行熒光素酶報告基因的檢測。結果顯示，DNA-PKcs沉默后，GFP-Aire/RAW細胞內的TLR1, TLR3, TLR8的啟動子轉錄活性明顯降低。結果表明，在RAW264.7細胞內，DNA-PKcs能夠協(xié)同Aire作用調控TLR1, TLR3和TLR8的表達。 3探討沉默DNA-PKcs后對小鼠腹腔巨噬細胞內Aire調控TLRs表達的影響 為了進一步研究在小鼠腹腔巨噬細胞內DNA-PKcs對Aire調控TLRs的表達是否有影響，沉默DNA-PKcs后，我們采用RT-qPCR的方法檢測TLRs的表達。結果顯示，在pEGFPC1/Aire瞬時轉染的細胞中，DNA-PKcs沉默后，TLR1,TLR3和TLR8的表達明顯下調。結果表明，在巨噬細胞內，DNA-PKcs能夠協(xié)同Aire作用調控TLR1, TLR3和TLR8的表達。 本研究證明了在小鼠巨噬細胞內DNA-PKcs可協(xié)同Aire作用調控TLR1、3、8的表達，為闡明Aire調控TLR1、3、8表達的機制、進而影響對病原微生物和自身衰老病變細胞的識別提供實驗依據(jù)，從而為Aire在外周表達的功能和意義提供了新的線索。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392

【共引文獻】

相關博士學位論文 前1條

1 朱武飛;自身免疫調節(jié)因子對巨噬細胞TLRs表達及其活化類型的影響[D];吉林大學;2011年

本文編號：1374097