類別:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/127
2024-03-05T18:17:32Z黏膜B細胞誘發調節性T細胞緩解過敏性呼吸道發炎之研究
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66157
標題: 黏膜B細胞誘發調節性T細胞緩解過敏性呼吸道發炎之研究; Regulatory T Cells Induced by Mucosal B Cells Alleviate Allergic Airway Hypersensitivity
作者: Kuan-Hua Chu; 朱冠驊
摘要: 氣喘是一種常見的慢性呼吸道發炎疾病。透過黏膜給予過敏原引起的免疫耐受性,已經被用來當做治療過敏疾病的方法。研究發現,皮耶氏體中最主要的細胞群─B細胞,可以促使調節性T細胞的生成。本篇研究即是針對皮耶氏體的B細胞,在產生調節性T細胞中扮演的角色,以及這些調節性T細胞的功能。利用體外抑制細胞增生實驗及測量細胞激素方法來確定這些由皮耶氏體B細胞刺激產生的調節性T細胞〈在此稱為Treg-of-B(P)細胞〉。對於測量 Treg-of-B(P)細胞的生理功能,我們利用細胞移植實驗將Treg-of-B(P)細胞注入用OVA蛋白致敏的小鼠體內,藉由偵測肺沖洗液中OVA特異性免疫球E的濃度,第二型T細胞激素,嗜伊紅性白血球增多現象,以及組織切片染色法確定Treg-of-B(P)細胞療效。
首先,我們證明連續五天口服餵食小鼠OVA 0.5毫克,可以降低肺沖洗液中第二型T細胞反應。我們也發現,高劑量的OVA〈20毫克〉會降低CD4 T細胞的數量。反之,低劑量餵食過敏原可以幫助生成調節性T細胞。試管外實驗證明Treg-of-B細胞具有抑制T細胞增生的能力。相較於脾臟B細胞,從皮耶氏體分出來的B細胞,在接觸過抗原之後,呈現較不活化的狀態。此外,藉由近距離的細胞接觸,以及介白素-10分泌促成此Treg-of-B細胞的抑制作用。利用螢光流式細胞儀分析發現,Treg-of-B(P)細胞會表現CTLA4、ICOS、OX40、PD-1和TNFRII。分泌的細胞激素方面,Treg-of-B(P)細胞會分泌低量的介白素-2,高量的介白素-10,甚至比自然產生的調節性T細胞還高。我們進一步的分析發現,介白素-10,GITR及PD-1都參與調控Treg-of-B(P)細胞的抑制作用中。除此之外,本篇實驗也比較了不同B細胞產生調節性T細胞的能力。在脾臟及皮耶氏體中的FOB及MZB細胞,可以將初始T細胞(naïve T cell)轉變為調節性T細胞。並且,皮耶氏體中的B細胞比起一般認為的專業的抗原呈現細胞,在此為樹突細胞,在生成調節性T細胞的能力更為明顯。為了確定此Treg-of-B(P)細胞的生理活性,我們將此調節細胞打入氣喘小鼠體內,發現不論在小鼠致敏前或致敏後打入細胞,都可以有效的抑制第二型T細胞激素產生,嗜伊紅性細胞浸潤,及緩解呼吸道阻力的症狀。
總合上述的發現,本篇研究證明在腸胃道的B細胞具有生成調節性T細胞的能力,並且藉此參與構成口服耐受性特性的過程,進而緩解氣喘疾病的症狀。; Asthma is one of the most common chronic airway inflammatory diseases. Induction of immunologic tolerance via mucosa has been used for treating allergic diseases. B cells, the major cell population in Peyer’s patches, have been shown to induce the development of Treg cells. This study aimed to investigate the role of B cells in Peyer’s patches to tolerance induction and on Treg cell functions. In vitro suppressive assay and ELISA were used to evaluate the function of T cells stimulated by Peyer’s patches B cells (Treg-of-B(P) cells). The therapeutic potential of Treg-of-B(P) cells on ovalbumin (OVA)-induced murine asthma is evaluated by measuring OVA-specific IgE, Th2 cytokine production, eosinophilia in Bronchoalveolar lavage fluid (BALF) and histopathological analysis.
First, we demonstrated that oral fed mice with OVA 0.5 mg for five days could diminish Th2 responses in BALF. High dose antigen administration (20mg) results in decreased CD4 T cell number, in contrast, low dose antigen helped Foxp3+ or IL-10 producing T cells induction. In vitro assay showed that Treg-of-B cells were found to exert suppressive function on T cell proliferation. Antigen-loaded B cells isolated from Peyer’s patches were more tolerogenic, compared with splenic B cells, and had the potential to generate more suppressive Treg-of-B cells via IL-10 production and cell-cell contract. Treg-of-B cells expressed CTLA4, ICOS, OX40, PD-1, and TNFRII and produced lower IL-2 and higher IL-10. Blocking experiment shows that IL-10, GITR and PD-1 mediate the suppressive effect of Treg-of-B(P) cells. Furthermore, we identify that FOB and MZB cells are capable of Treg cells generation. In addition, Peyer’s patches B cells are much better than Peyer’s patches DCs on generating Treg cells. In the murine model of asthma, an adoptive transfer of Treg-of-B(P) cells pre- or post- immunization could sufficiently suppress Th2 cytokine production and eosinophil infiltration, and alleviate asthmatic symptoms.
B cells isolated from gut-associated lymphoid tissues, GALT, can generate regulatory T cells that may be important in oral tolerance and be applicable for alleviating allergic symptoms.2012-01-01T00:00:00Z間葉幹細胞應用在改善過敏性呼吸道重塑的機制探討
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61749
標題: 間葉幹細胞應用在改善過敏性呼吸道重塑的機制探討; Study on the Modulatory Effects of Mesenchymal Stem Cells in the Pathogenesis of Allergic Airway Remodeling
作者: Yung-Ting Chen; 陳詠婷
摘要: 氣喘是常見的呼吸道過敏性疾病,近幾年有逐漸增加的趨勢。目前已知過敏性氣喘是由第二型T輔助型細胞所引起,伴隨著呼吸道過度反應、嗜酸性球浸潤以及氣道重塑 (airway remodeling) 等特徵。氣道重塑為呼吸道因慢性發炎產生結構性改變的情形,包括:表皮細胞、杯狀細胞及呼吸道平滑肌增生、呼吸道黏液增加、呼吸道纖維化等情況。來自於骨髓的間葉幹細胞 (mesenchymal stem cells, MSCs),屬於多潛能性幹細胞,是一種能自我更新、繁殖並能分化成不同種類的組織的細胞。同時,近幾年也證實間葉幹細胞具有免疫調節的功能。我們利用卵清蛋白 (ovalbumin,OVA) 連續刺激小鼠呼吸道而建立小鼠呼吸道慢性發炎動物模式,探討間葉幹細胞是否能有效抑制發炎反應並減緩呼吸道重塑的情況。首先,我們從老鼠骨髓細胞培養出間葉幹細胞,經過繼代培養,確認間葉幹細胞表現抗原,並利用免疫細胞刺激劑 (ConcanaalinA, ConA) 測試間葉幹細胞其免疫抑制功能,當脾臟淋巴細胞與間葉幹細胞共同培養時,間葉幹細胞能有效抑制其增生。同時,我們利用不同的培養條件以及處理細胞的方法,測試間葉幹細胞是否能有效抑制纖維母細胞 (fibroblasts) 合成不同型的膠原蛋白(collagens),結果發現當纖維母細胞及間葉幹細胞在沒有細胞與細胞接觸而共同培養的情況下,不同型的膠原蛋白有被抑制表現的情況。更進一步,我們利用尾巴靜脈注射方式將不同代數的間葉幹細胞打入呼吸道慢性發炎小鼠中,最後再給予鼻腔刺激OVA引發呼吸道發炎,探討間葉幹細胞的治療效果,及不同代數之間的療效。結果顯示,給予不同代數間葉幹細胞皆能有效減緩呼吸道過度反應,此外,也造成細胞浸潤狀況、肺泡灌洗液中的相關細胞激素以及黏液產生的狀況產生不同程度的改善。同時我們也發現在有打入間葉幹細胞的小鼠中,嗜中性球的數量以及與第十七型輔助型T細胞 (Th17)相關激素皆明顯上升。未來將更進一步探討間葉幹細胞在免疫抑制上的作用機制及其確切的作用位置; Asthma is a common allergic disease and its prevalence has been increased gradually. Asthma is characterized by induction of Th-2 cells, airway hyperresponsiveness, eosinophil infiltration and airway remodeling. Characteristic structural changes of airway remodeling include epithelial cell mucus metaplasia, smooth muscle hypertrophy, subepithelial fibrosis, and increased angiogenesis. Bone marrow derived mesenchymal stem cells (MSCs) are a self-renewing population of multipotent stem cells and also have been recently demonstrated to suppress harmful immune responses. Here we established an OVA-sensitized animal model of asthmatic airway remodeling to investigate whether MSCs could alleviate OVA-induced airway inflammation and attenuate airway remodeling progression. First, MSCs were isolated from mouse bone marrow, characterized by their phenotypes and immunosuppressive function in vitro. Also, we used different culture conditions to investigate whether MSCs could suppress collagen synthesis of fibroblast in vitro. And the results indicated that MSCs could not only exert inhibitory effect on the proliferation of lymphocytes under ConA stimulation but also suppress different types of collagen expression in transwell assay. Furthermore, to determine the therapeutic effects of different passages of MSCs, different passages of MSCs were injected intravenously before OVA challenged respectively into chronic animal models of airway inflammation. And we found airway hyperresponsiveness, eosinophil infiltration, cytokine level in bronchoalveolar lavage fluid, and mucus production in airway were attenuated after administration of MSCs. In addition, we also found that netrophil infiltration and Th-17 associated cytokine, IL-17, were significantly increased after MSCs administration. In the future, it will be interesting to clarify the actual immuomodulatory mechanism and action sites of MSCs.2012-01-01T00:00:00Z間葉幹細胞分泌之基質金屬蛋白酶-9在其免疫調節功能之角色
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53724
標題: 間葉幹細胞分泌之基質金屬蛋白酶-9在其免疫調節功能之角色; The role of mesenchymal stem cell-derived matrix metalloproteinase 9 (MMP-9) in immunomodulation
作者: Tzu-Yu Feng; 馮資喻
摘要: 骨髓間葉幹細胞(Mesenchymal stem cells, MSCs)為多潛能性幹細胞,具有自我更新、繁殖及分化成不同細胞的能力。因著眾多試管內及體內試驗證明間葉幹細胞具有優秀的免疫調節功能,將間葉幹細胞應用在治療免疫發炎反應失調疾病被視為相當有潛力的替代療法。然而將間葉幹細胞推展至臨床使用的過程並不順利。儘管有些人體試驗結果十分成功,但有些人體臨床試驗則顯示間葉幹細胞對發炎並無抑制功能。因此,為了間葉幹細胞的實用化,進一步探討並優化其免疫調節功能實屬必要。
近年來發現間葉幹細胞的免疫調節功能部分會被周邊發炎環境所調控。過去,本實驗室發現間葉幹細胞會分泌高量的基質金屬蛋白酶-9(Matrix metalloproteinase 9, MMP-9)這個在許多發炎疾病中皆會被高度表現的蛋白。本論文延續過去的基礎,進一步探討發炎因子刺激是否會促使間葉幹細胞表現更高量的MMP-9,以及MMP-9是否會影響間葉幹細胞表現細胞激素(Cytokine)與趨化激素(Chemokine)的功能。我們發現相較於干擾素γ (Interferon-γ, IFN-γ)刺激,經脂多醣(Lipopolysaccharide, LPS)及腫瘤壞死因子α (Tumor-necrosis factor α, TNF-α)刺激後的間葉幹細胞會表現較高量的MMP-9。並且抑制MMP-9會降低間葉幹細胞表現介白素-6(Interleukin-6, IL-6)及干擾素誘導蛋白-10(C-X-C motif chemokine 10/Interferon-γ-induced protein 10, CXCL10/IP-10)的能力。
另外,抑制T細胞增生及誘導調節性T細胞(Regulatory T cell, Treg)產生為間葉幹細胞抑制免疫反應的兩大利器。本論文亦探討經過不同發炎因子刺激後的間葉幹細胞是否會因著分泌MMP-9表現量之不同而改變其抑制T細胞增生及誘導調節性T細胞的能力。實驗結果證實MMP-9確有改變間葉幹細胞對T細胞的免疫調節功能。而抑制MMP-9會經由降低IFN-γ與介白素-1β (Interleukin-1β, IL-1β)及增加CD44在T細胞的表現量,因而增加間葉幹細胞對T 細胞的抑制功能及誘導調節性T細胞產生的能力。; Bone marrow-derived mesenchymal stem cells (MSC) possess pluripotent and self-renewal properties. Importantly, MSC exhibit potent immunomodulatory abilities both in vitro and in vivo. Based on these findings, transplantation of MSC is considered as a promising therapy for several inflammatory diseases. However, several clinical trials have demonstrated conflicting results. To realize the potential of MSC therapy, it is necessary to investigate the approaches to optimize MSC immunomodulation.
Recently, several studies indicate that the immunomodulatory abilities of MSC are partially changed by inflammatory stimuli. In our previous study, MMP-9 was highly expressed by MSC. Based on these information, this study aimed to examine that inflammatory mediators stimulation would change the MMP-9 expression in MSC or not. Then, we also investigated the role of MSCs-derived MMP-9 in the cytokines and chemkines production process. The results showed that lipopolysaccharide (LPS)-primed and Tumor-necrosis factor α (TNF-α)-primed MSC (MSC1 and MSCα) expressed higher MMP-9 than interferon-γ (IFN-γ)-primed MSC (MSCγ). Furthermore, blockade MMP-9 significantly reduced the levels of Interleukin-6 (IL-6) and C-X-C motif chemokine 10/Interferon-γ-induced protein 10 (CXCL10/IP-10) in MSCs.
Suppression of T cells proliferation and induction of regulatory T cells (Treg) are two important mechanism underlying the MSC immunomodulation. We also investigated the influences of MMP-9 on these two important functions of MSC. In this section, we found that inhibition of MMP-9 significantly improved the ability of MSC1 and MSCα to suppress T cells proliferation and induce Treg.2015-01-01T00:00:00Z鑑定蛋白酪胺酸磷酸酶(非受體二十二型)之細胞核定位訊號
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45668
標題: 鑑定蛋白酪胺酸磷酸酶(非受體二十二型)之細胞核定位訊號; Identification of Nuclear Localization Signal (NLS) in PTPN22
作者: Yi-Wei Sun; 孫宜緯
摘要: 蛋白酪胺酸磷酸酶非受體二十二型為在細胞質中負向調控T細胞訊息路徑的酪胺酸磷酸酶。我們實驗室近來在細胞核中發現此酵素。然而,蛋白酪胺酸磷酸酶非受體二十二型進入細胞核的機制仍未明。先前已知帶有細胞核定位訊號的蛋白質可以被運送進細胞核中。我們證實蛋白酪胺酸磷酸脢非受體二十二型的區域C為潛在的細胞核定位訊號,而且區域C對於細胞核運送是必須存在的。帶有區域C被刪除的蛋白酪胺酸磷酸脢非受體二十二型之螢光表現質體在轉染細胞後,發現其螢光無法分布於細胞核中。從點突變實驗中,我們也證實蛋白酪胺酸磷酸脢非受體二十二型的A和B點突變之後,將會損壞此蛋白質進入細胞核的能力。進一步研究負責運送蛋白酪胺酸磷酸脢非受體二十二型進入細胞質的胺基酸序列之實驗中,我們將區域I或/和區域II和雙倍的綠色螢光蛋白克隆在一起,製造出融合蛋白,驗證這些區域對於將綠色螢光蛋白送運進入細胞核的能力為何。從實驗結果發現,蛋白酪胺酸磷酸酶非受體二十二型的區域I能將綠色螢光蛋白運送進入細胞核。我們的實驗指出蛋白酪胺酸磷酸酶非受體二十二型的區域I為細胞核定位訊號且在運送蛋白質進入細胞核中扮演十分重要的角色。; PTPN22 is a protein tyrosine phosphatase that participates in the negative regulation of T cell signaling in cytoplasm. Recently, our lab shows that PTPN22 could locate inside the nucleus. However, the molecular mechanism of PTPN22 in nuclear import is poorly understood. Proteins that import from the cytosol to the nucleus contain basic amino acids-rich nucleus localization signal (NLS). We demonstrated Region C of PTPN22 was a potential NLS and necessary for nuclear import. Cells transfected with fluorescent vectors encoding Region C-deleted PTPN22 showed clear nuclear distribution. Mutation analysis showed A and B mutations of PTPN22 are critical sites for nuclear import of PTPN22, because the Mutant A and B impaired the capability of nuclear transport of PTPN22. To further identify sequences responsible for nuclear import of PTPN22, Region I or/and Region II was fused with double GFP and examined their abilities to target GFP into the nucleus. The results showed that Region I of PTPN22 could import GFP into the nucleus. Our data indicated that the Region I of PTPN22 served as NLS and played a vital role in nuclear import.2010-01-01T00:00:00Z