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標題: | 代謝酵素Atp5d和Cox6a2調控樹突細胞的發育 Metabolic enzymes Atp5d and Cox6a2 Regulate Dendritic Cell Development |
作者: | Yen-Yu Lin 林彥佑 |
指導教授: | 李建國(Chien-Kuo Lee) |
關鍵字: | 代謝酵素,樹突細胞,發育,哺乳動物雷帕黴素靶蛋白,AMP依賴蛋白激?, Metabolism,dendritic cell,development,mTOR,AMPK,Atp5d,Cox6a2, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 樹突細胞 (DC),包括漿狀樹突細胞(pDC)及傳統樹突細胞(cDC)對於調節免疫反應十分重要。傳統樹突細胞呈現抗原給T細胞是重要的,然而漿狀樹突細胞卻是產生I型干擾素為主的細胞以適應發炎和感染,這兩者都有助於宿主抵禦病毒感染或癌症發生。在樹突細胞成熟和用TLR刺激活化後,會用AKT-,HIF1-α-或mTOR所依賴的訊息傳導途徑過程去增加醣酵解作用和脂肪酸合成。然而,前驅細胞在發育成為熟樹突細胞的代謝途徑變化仍然是不清楚。這裏我們認為從前驅細胞發育成pDC和cDC同樣會需要不同的代謝過程。在體外利用Lin-Flt3 +(LF)前驅細胞的去培養DC時我們加入Compound C(AMPK的抑制劑)會降低cDC並增加pDC發育,但如果使用Rapamycin(mTOR的抑制劑),或Ly294002(Akt的抑制劑)則增加cDC但減少pDC的發育。此外,LF衍生的cDC(LF-cDC)表現出較低的線粒體質量,但與LF衍生的pDC (LF-pDC)相比則產生更高的細胞ATP。有趣的是在生物能量分析中發現,LF-cDC具有更高的OCR和ECAR,並且比LF-pDC具有更高的能粒線體活性。這些結果顯示AMPK和mTOR訊息傳導途徑對pDC和cDC的發育具有相反的作用,並且pDC和cDC具有不同的生物能量學特徵。生物資訊學的分析顯示,61個代謝酶的基因表現量在pDC與cDC中各有高低。我們使用一株前期永生細胞株(immortalized hematopoietic stem and progenitor, iHSPC)做基因靜默61種代謝酶的候選基因,結果發現一組氧化磷酸化相關酶的基因會影響pDC發育。其中,Atp5d和Cox6a2似乎扮演非常關鍵角色,同時任一基因的基因靜默都導致在iHSPC中pDC發育的嚴重減少。有趣的是, Cox6a2在pDC中基因表現量高於cDC和其他免疫細胞。此外,Cox6a2基因靜默還增加了iHSPCs中基礎和抗黴素A (Antimycin A)所誘導的線粒體ROS和基礎ECAR和OCR的測量。更有趣的是,Flt3L和TLR7所依賴的mTOR磷酸化也被阻斷,使得線粒體活性與mTOR功能和pDC發育有一定相關聯。這些結果顯示,新陳代謝確實可以控制DC的發育。未來我們將需要進一步的實驗來揭示有可能的機制。 Dendritic cells (DCs), including plasmacytoid DC (pDC) and conventional DC (cDC) are crucial for regulating the immune response. While cDCs are important for presenting antigens to T cells, pDCs mainly produce type I interferon in response to inflammation and infection, both of which help the hosts defend against viral infections or cancer. During DC maturation and activation with TLR agonists, glycolysis and fatty acid synthesis are increased through AKT-, HIF1-α- or mTOR-dependent signaling pathways. However, the requirement of metabolic changes in progenitor cells to develop into mature DC remains elusive. We hypothesize that development of pDC and cDC from progenitors requires differential metabolic profiles. While compound C, an inhibitor of AMPK, decreased cDC but increased pDC development, rapamycin and Ly294002, inhibitors of mTOR, otherwise increased cDC but decreased pDC development from the Lin-Flt3+ (LF) progenitors. Moreover, LF-derived cDC (LF-cDC) exhibited lower mitochondrial mass but product higher cellular ATP compared to LF-derived pDC (LF-pDC). Interesting, bioenergetics analysis showed that LF-cDC had higher OCR and ECAR and were more energetic active than LF-pDC. These results suggest that AMPK and mTOR pathways have opposite effects on pDC and cDC development and that pDC and cDC have different bioenergetics profiles. In silico analysis revealed that 61 genes that encode metabolic enzymes are differentially expressed in pDCs versus cDCs. Knockdown of the candidate genes encoding the 61 metabolic enzymes in an immortalized hematopoietic stem and progenitor (iHSPC) cell line showed that gene targeting oxidative phosphorylation-related enzymes affected pDC development. Among them, Atp5d and Cox6a2 appear to be very crucial. As knockdown of either gene resulted in severe impairment of pDC development from the iHSPCs. Interestingly, Cox6a2, a cytochrom c oxidase subunit, is highly expressed in pDC compared to cDC and other immune cells. Moreover, Cox6a2 knockdown also increased basal and antimycin A-induced mitochondrial ROS and basal ECAR and OCR in iHSPCs. Interestingly, Flt3L and TLR7-dependent mTOR phosphorylation was blocked, linking the mitochondria activity to mTOR function and pDC development. The results indicate that metabolism can indeed control DC development. Further experiments will be required to unveil the underlying mechanisms |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70757 |
DOI: | 10.6342/NTU201802730 |
全文授權: | 有償授權 |
顯示於系所單位: | 免疫學研究所 |
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