類別:http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/1202024-03-28T09:15:57Z2024-03-28T09:15:57Z齒源性角化囊腫及含齒囊腫中之蘭格罕氏細胞數目及其與臨床病理參數之相關性Chun-Han Chang張君涵http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/203382021-06-08T02:45:36Z2017-01-01T00:00:00Z標題: 齒源性角化囊腫及含齒囊腫中之蘭格罕氏細胞數目及其與臨床病理參數之相關性; Langerhans cell counts in odontogenic keratocysts and dentigerous cysts and their correlations with clinicopathological parameters
作者: Chun-Han Chang; 張君涵
摘要: 背景:蘭格罕氏細胞是主要存在於上皮內的抗原呈遞細胞,本研究評估齒源性角化囊腫(OKC)及含齒囊腫(DC)中之蘭格罕氏細胞(LC)數目。
方法:本研究主要利用anti-CD1a及anti-S100蛋白質免疫組織化學染色法,評估於60例OKC標本及80例DC標本中之蘭格罕氏細胞數目及其與臨床病理參數之相關性。於60例OKC標本中,評估60個無發炎部位,39個輕度或中度發炎部位,13個重度發炎部位之蘭格罕氏細胞數目;於80例DC標本中,評估80個無發炎部位,33個輕度或中度發炎部位,9個重度發炎部位之蘭格罕氏細胞數目。
結果:60例OKC樣本中,在內襯上皮和上皮下方結締組織中之平均CD1a陽性或S100陽性之LC細胞數目,從無發炎部位至輕度/中度發炎部位,及至重度發炎部位,其蘭格罕氏細胞數目皆逐步顯著增加(P值皆< 0.001)。有發炎之OKC部位比無發炎之OKC部位,具有較厚之內襯上皮。此外,在厚內襯上皮(> 100 μm)組之OKCs中,內襯上皮和上皮下結締組織中之平均CD1a陽性或S100陽性之LC細胞數目,皆明顯高於薄內襯上皮(≤ 100 μm)組之OKCs (P值皆< 0.001)。於80例DC樣本中,也可發現在內襯上皮和上皮下方結締組織中之平均CD1a陽性或S100陽性之LC細胞數目,從無發炎部位至輕度/中度發炎部位,及至重度發炎部位,其蘭格罕氏細胞數目皆逐步顯著增加(P值皆< 0.001)。有發炎之DC部位比無發炎之DC部位,具有較厚之內襯上皮。此外,在厚內襯上皮(> 50 μm)組之DCs中,內襯上皮和上皮下結締組織中之平均CD1a陽性或S100陽性之LC細胞數目,皆明顯高於薄內襯上皮(≤ 50 μm)組之DCs (P值皆< 0.001)。
結論:本研究發現發炎之等級與OKC中之LC數量,有顯著相關。在無發炎OKC的內襯上皮中,只可發現極少量之LC,此表示OKC內襯上皮細胞之免疫監視能力明顯喪失,這可解釋為什麼OKC具有強大臨床侵犯性、強大細胞增殖潛力和高的復發率。此外在DC研究中,LC數量之增加與高發炎程度和厚內襯上皮有顯著相關。在無發炎DC之內襯上皮中,只發現極少數之LC,也表明DC患者之內襯上皮細胞免疫監視能力明顯下降。然而,需要進一步研究來確認上皮免疫監視能力之降低和DC之囊腫增大是否有關。; Background/Purpose: Langerhans cells (LCs) are antigen-presenting cells that reside mainly within the epithelium. This study assessed the LC counts in odontogenic keratocysts (OKCs) and dentigerous cysts (DCs).
Methods: This study used anti-CD1a and anti-S100 protein immunostains to assess the LC counts in 60 OKCs and 80 DCs and the correlations of LC counts with clinicopathological parameters of 60 OKCs and 80 DCs. The LC numbers in the lining epithelia and subepithelial connective tissues were counted at 60 OKC sites without inflammation, 39 OKC sites with mild/moderate inflammation, and 13 OKC sites with severe inflammation from 60 OKC specimens as well as at 80 DC sites without inflammation, 33 DC sites with mild/moderate inflammation, and 9 DC sites with severe inflammation from 80 DC specimens.
Results: For 60 OKC cases, the mean CD1a-positive or S100-positive LC counts in the lining epithelia and subepithelial connective tissues increased significantly from no inflammation through mild/moderate inflammation to severe inflammation OKC sites (all P-values < 0.001). OKC sites with inflammation had thicker lining epithelia than those without inflammation. Moreover, the mean CD1a-positive or S100-positive LC counts in the lining epithelia and subepithelial connective tissues of OKCs were significantly higher in the thicker lining epithelium (> 100 μm) group than in the thinner lining epithelium (≦ 100 μm) group (both P-values < 0.001). For 80 DC cases, the mean CD1a-positive or S100-positive LC counts in the lining epithelia and subepithelial connective tissues also increased significantly from no inflammation through mild/moderate inflammation to severe inflammation DC sites (all P-values < 0.001). DC sites with inflammation had thicker lining epithelia than those without inflammation. Moreover, the CD1a-positive or S100-positive mean LC counts in the lining epithelia and subepithelial connective tissues of DCs were significantly higher in the thicker lining epithelium (> 50 μm) group than in the thinner lining epithelium (≦ 50 μm) group (both P-values < 0.001).
Conclusion: A significant association of inflammation grade with the number of LCs in OKCs is found in this study. The finding of scare LCs in the lining epithelia of OKCs without inflammation indicates the loss of immunosurveillance ability against the OKC lining epithelial cells; this can explain why OKCs have aggressive clinical behavior, a great growth potential, and a high recurrence rate. Moreover, the increased LC number in DCs is also associated with high-grade inflammation and thick lining epithelium. The finding of few LCs in the lining epithelia of DCs without inflammation also indicates the reduced immunosurveillance ability against DC lining epithelial cells in DC patients. However, it needs further studies to confirm the role of reduced lining epithelial cell immunosurveillance in the enlargement of the DC.2017-01-01T00:00:00Z高濃度葡萄糖藉由水解磷酸脂促進前列腺癌細胞淋巴管生成因子Ya-Jan Yang楊雅然http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/525972021-06-15T16:19:53Z2015-01-01T00:00:00Z標題: 高濃度葡萄糖藉由水解磷酸脂促進前列腺癌細胞淋巴管生成因子; High Glucose Induces Vascular Endothelial Growth Factor-C through Lysophosphatidic Acid Signals in Human Prostate Cancer PC-3 Cells
作者: Ya-Jan Yang; 楊雅然
摘要: 前列腺癌 (prostate cancer) 是男性常見的癌症之一,在台灣男性癌症中死亡率排行第七位,常見病徵有淋巴和骨髓轉移等現象。而在第二型糖尿病患者中,經由臨床統計發現其具較高的罹癌機率,高血糖會促進有氧糖解,增進癌細胞生長速度,但目前的研究對於高血糖在前列腺癌的影響尚具爭議。水解磷酸脂 (Lysophosphatidic acid, LPA) 為細胞膜上衍生出的小分子傳訊脂質,由溶血磷脂醯膽鹼 (Lysophosphatidylcholine, LPC) 在自分泌運動因子 (Autotaxin, ATX) 酵素的作用下切除膽鹼而形成。已知LPA會和細胞膜上的水解磷酸脂受器 (LPA receptors)結合,促進癌細胞生長、移動和附著。在實驗室先前的研究中發現,在前列腺癌PC-3細胞株中,LPA可透過水解磷酸脂受器1 (LPA1) 和水解磷酸脂受器3 (LPA3) 促進淋巴管新生因子C型 (Vascular endothelial growth factor-C, VEGF-C) 的表現,進而影響癌症的淋巴轉移;此外,在糖尿病患者及老鼠中,也發現 ATX 的表現量增加。因此本實驗的目的探討在前列腺癌PC-3細胞株中,高濃度葡萄糖是否藉由水解磷酸脂訊號傳遞路徑促進有氧糖解和淋巴管新生因子C型的表現,導致前列腺癌的惡化。實驗結果顯示10 mM和20 mM的高濃度葡萄糖會增加VEGF-C 和ATX 的核醣核酸和蛋白質的表現。並透過抑制性藥物和基因敲除 (knockdown) 的實驗證實高濃度的葡萄糖確實透過ATX、LPA1/3、過氧化物 (Reactive oxygen species, ROS) 和晶狀體上皮源性生長因子 (Lens epithelium-derived growth factor, LEDGF) 的訊息傳遞路徑促進VEGF-C的表現。此外,我們也發現高濃度葡萄糖會增加鈣網蛋白 (Calreticulin, CRT) 的表現,並藉由基因knockdown的實驗證實CRT會調控ATX的表現。另一方面,由海馬生物能量測定儀結果顯示高濃度葡萄糖和LPA會提高有氧糖解的速率,反之,加入抑制LPA1/3藥物後有氧糖解速率下降。由這些結果證實,不正常的葡萄糖代謝會促進ATX的表現並增加LPA的含量,進而促進前列腺癌的淋巴管新生,造成前列腺癌病患的惡化。; Prostate cancer is one of the most frequently diagnosed cancers in males and usually metastasizes to various organs but particularly to local lymph nodes. Clinical evidences suggest that type II diabetes mellitus has been known to increase the risk of several cancers. Hyperglycemia would increase aerobic glycolysis and promote cancer growth, but the effects on prostate cancer are still controversial. Lysophosphatidic acid (LPA) is a small glycophospholipid that mediates multiple behaviors by activating LPA receptors in cancer cells, such as cell proliferation, migration and adhesion. In our previous studies, LPA could enhance VEGF-C expression through activating LPA receptor 1/3 in prostate cancer. On the other hand, autotaxin (ATX), an enzyme responsible for LPA synthesis, was up-regulated in diabetic patients and mice. In this study, we used PC-3 cell line as a model to investigate whether high glucose induced lymphangiogenesis and aerobic glycolysis through LPA signals to drive cancer progression. Our results demonstrated that the mRNA and protein expression levels of VEGF-C and ATX were increased after 10 mM and 20 mM high glucose treatments in PC-3 cells. By pharmacological blockers and knockdown experiments, we confirmed that the expression of VEGF-C was mediated through ATX, LPA1/3, ROS and LEDGF dependent pathways. Furthermore, the mRNA and protein levels of calreticulin (CRT) were up-regulated under high glucose conditions. By knockdown experiments, we demonstrated that ATX might be a downstream signal of CRT. On the other hand, high glucose and LPA treatments also enhanced the aerobic glycolysis. Taken together, these results suggested that the abnormal glucose metabolism might lead to LPA synthesis and therefore the subsequent pathological conditions of prostate cancer. These novel findings could potentially provide new strategies for prostate cancer treatments.2015-01-01T00:00:00Z電壓感應開啟鉀離子通道與蜘蛛毒蛋白間的動力學研究Yu-Wen Shih石又文http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/353002021-06-13T06:47:16Z2005-01-01T00:00:00Z標題: 電壓感應開啟鉀離子通道與蜘蛛毒蛋白間的動力學研究; Kinetic study of interactions between voltage-gated K+-channels and toxins from spider venom
作者: Yu-Wen Shih; 石又文
摘要: 電壓感應開啟式鉀離子通道(Voltage-gated potassium channel, Kv channel)普遍存在於各類組織,主要負責在細胞膜興奮時開啟,造成再極化。通常由四個次單元體組成,每個次單元體上含有通道形成區域(pore-forming domain)及四個電壓感受過膜片段(voltage-sensing transmembrane segments),稱之為S1-S4。其中S4是最主要的電壓感受體(voltage sensor)。又目前已知S3的C端(S3C)可與通道開啟調整毒素,如蜘蛛毒蛋白(Hanatoxin1, HaTx1;Stromatoxin1, ScTx1)作用使通道開啟時所需電壓改變。而最常被用來研究S3C 結構-功能之電壓感應開啟式鉀離子通道Kv 2.1 channel(drk1)屬shab family,會受蜘蛛毒蛋白作用而不易開啟;相對地,shaker(Kv1.1 channel)則不受蜘蛛毒蛋白之影響。S3C 可能之二級結構也因此透過相關研究而有逐步瞭解,且目前一般相信S3C含有一段獨立的α-helix。
經由本實驗室前人對電壓感應開啟式鉀離子通道S3C與蜘蛛毒蛋白所做三度空間結構上的分析,及利用分子模擬與嵌合的技術,可得到電壓感應開啟式鉀離子通道S3C與蜘蛛毒蛋白交互作用之分子模型。近幾年,由Roderick Mackinnon所帶領的研究團隊對於電壓感應開啟式鉀離子通道之電壓感受體的研究有一些異於傳統之突破。他們利用古溫泉細菌Aeropyrum pernix之電壓感應開啟式鉀離子通道(KvAP)的蛋白質單晶,以X光繞射得到結構及配合一系列與電壓感受體相關的電生理實驗研究,提出不同於傳統Kv channel結構之模型,並且利用序列同源性比對,將此一模型推廣適用到大部分真核生物之電壓感應開啟式鉀離子通道上。此模型與之前的許多真核生物之電壓感應開啟式鉀離子通道的電生理實驗結果未盡相符,亦與本實驗室前人所做之作用機制模型的基礎假設有所差異。
因此,在本論文中,筆者以之前本實驗室前人所完成的關於Kv2.1 S3C與Hanatoxin結合時之模擬計算為基礎,透過停止流螢光光譜(stopped-flow),對S3C片段與Hanatoxin的作用,進行了一系列的實驗分析與探討。所得到的生化動力學數據,可以作為檢驗先前提出的分子機制及探討S3C片段之生理可能構形時之重要依據。配合上先前之模擬計算,我們也對此一作用發生時各分子在細胞膜上空間分佈的關係作了深入的推理。
透過Hanatoxin或Stromatoxin與Kv2.1 S3C片段結合的動力學實驗,可以計算出結合發生時之kon、koff值。比較了使用Kv1.1和Kv2.1 S3C 變異株作為控制組的結果後,若再配合加入TFE(2,2,2-trifluoroethanol)使Kv2.1 S3C片段重新摺疊(refolding),可以清楚得知, Kv2.1 S3C片段與Hanatoxin之結合可能確實依照先前分子模擬預測之機制進行。至於Kv2.1 S3C不同變異株(hydrophobic variant/hydrophilic variant)之間的比較部分,我們則得到稍異於分子模擬預測之結果:疏水性與親水性的作用應同等重要,突變任一類鍵結所需殘基均能使蜘蛛毒蛋白與Kv2.1 S3C 片段之結合能力消失,而非如之前預測的由極性反應擔負較重要之角色。綜合以上生化動力學實驗並配合分子模擬預測之結果,加上先前諸多電生理的資料,我們推測負責與Hanatoxin鍵結之Kv2.1 S3C片段殘基應位於細胞膜邊界處,靠近磷脂(phospholipids)之hydrophilic head處。因此整個Kv2.1 S3C 片段必須以稍稍傾斜的方式坐落於細胞膜之外緣。因此傳統的結構模型,由於指出了external crevice的特性,似乎較符合此處的整體空間關係。至於Stromatoxin,我們認為在序列上與Hanatoxin十分接近,並且同為抑制Kv2.1 channel之毒蛋白,故應可得到類似於Hanatoxin之生化動力學實驗結果;但實際上所得之數據卻傾向以迥異於對Hanatoxin之分析來詮釋。由於Stromatoxin是一種近年來新發現之蜘蛛毒蛋白,功能測試之數據並不完整,僅知其對於Kv2.1擁有如Hanatoxin一般使通道趨於不易開啟之類似特性,其抑制Kv2.1 channel之詳細作用機制則仍不十分明瞭,但相信其細部摺疊在生理特性上應有重要意義。這些都有待更深入地研究以釐清其中的關鍵因素。
本論文不僅將先前關於蜘蛛毒與Kv通道反應機制之研究以生化實驗給予相當程度的驗證,也呈現了一些新且有趣的現象,有待更進一步的觀察與討論。; Voltage-gated potassium channels are found in a wide variety of tissues, where their primary role is to respond to the membrane excitation to allow the repolarization phase of an action potential to occur and therefore the K+ ions can efflux. Such channels are normally homotetramers and each subunit contains four voltage-sensing transmembrane segments, namely S1 through S4, whereas S5 and S6 form the pore. Among them, S4 may play the most crucial role in sensing the voltage change. Kv2.1, a member of shab potassium channel family, is one the most commonly applied channels in study of the structural-functional correlation for voltage sensing. Upon binding of hanatoxin, the midpoint of the curve for required gating potential of Kv2.1 can be shifted to the right, which means more difficult to open the channels under the same condition. On the contrary, shaker potassium channels do not show such effect. The secondary structural arrangement of S3C has been, due to such studies, intensively analyzed and the existence of an independent α-helix was then suggested.
It has been accomplished to establish a model and hypothesis describing the molecular details of hanatoxin-binding induced gating. This was based on the 3-D structural analysis with molecular docking and simulations. Recently, research by Mackinnon’s group has led to certain controversial developments in the voltage-sensing theory of Kv channels. Crystal structure of KvAP channel from archaeum Aeropyrum pernix revealed, combined with a series of electrophysiological experiments and sequence comparisons, a novel ‘voltage-sensor paddle’ model, which was anticipated to be applied on eukaryotic Kv channels. However, such revolutionary idea did challenge the conventional “translocation” model and contradict to a few previously acknowledged experimental results. Such discovery has also brought gross impact on our proposed mechanism which was based upon the conventional translocation model in Kv channels.
Therefore, in this thesis, we have performed the kinetic analysis with stopped-flow to examine our previously proposed hypothesis. Such binding study, in combination with related calculations, provides further possibility to consider in a more decent way the discussion of the reasonable conformation and membrane distribution of S3C segment in the toxin-Kv channel interactions.
The binding rate constant kon and release rate constant koff for interactions between hanatoxin/stromatoxin and Kv2.1 S3C segments can be calculated through the kinetic analysis with stopped-flow. Upon utilization of Kv2.1 S3C mutants and Kv1.1 S3C as control experiments, together with the appropriate additions of trifluoroethanol in different concentrations, which allow the refolding of Kv2.1 S3C to occur, it has been indicated that binding of hanatoxin and Kv2.1 S3C may follow the molecular details described in our proposed mechanism. However, the comparison between the hydrophobic and hydrophilic interactions required for binding between hanatoxin and Kv2.1 S3C observed from rationally designed mutants (hydrophobic part v.s. polar part of residues) suggests that both types of interaction are equally crucial for binding. Mutation of either part of residues can result in the abolishment of binding ability for hanatoxin and Kv2.1 S3C. Polar interactions should not be the only dominant factor able to affect such binding as predicted thru simulation study. All together, it is reasonable to comprehend that the S3C residues required for binding with hanatoxin should be located at the boundary of cell membrane, nearby the hydrophilic heads of phospholipids (or interfacial area of external face) with a slight tilting angle. Therefore the conventional ‘translocation’ model may fulfill such requirement better, especially considering the spatial orientations of transmembrane segments around the external crevice. On the other hand, due to the sequence and functional similarity between stromatoxin and hanatoxin, we did expect that the kinetic data for stromatoxin present themselves in a very similar pattern as those of hanatoxin do. However, surprisingly, all the observations tend to draw our attention a totally different way of interpretation. Probably the structural details, rather than general structural feature, of stromatoxin may play pivotal role in the binding behavior. This awaits further investigations. This thesis provides experiments support on our previously proposed molecular mechanism of tarantula toxins binding induced gating in Kv channels to a certain extent. It also leads to some interesting questions open for future study.2005-01-01T00:00:00Z間葉幹細胞應用在類風濕性關節炎動物模式發炎改善之探討Ning-Ling Huang黃甯翎http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/653982021-06-16T23:40:40Z2012-01-01T00:00:00Z標題: 間葉幹細胞應用在類風濕性關節炎動物模式發炎改善之探討; Study on the Anti-inflammatory Effect of Mesenchymal Stem Cells in Murine Model of Collagen-Induced Arthritis
作者: Ning-Ling Huang; 黃甯翎
摘要: 研究背景:
類風濕性關節炎 (Rheumatoid arthritis, RA) 為常見的自體免疫疾病。目前已 知許多免疫細胞參與此疾病的病理機制。在疾病初期,T 淋巴細胞被認為是導致 疾病發生與影響病程重要因子之一。間葉幹細胞 (Mesenchymal stem cells, MSCs) 為多潛能性幹細胞
(pluripotent stem cells),可由骨髓或其他組織中取得,具有自 我更新、繁衍並且可分化成多種不同的細胞與組織。近年許多文獻報告指出間葉 幹細胞具有免疫調節的能力。本篇研究希望了解間葉幹細胞在治療類風濕性關節 炎動物模式中的應用性。
實驗方法:
從小鼠骨髓中培養出間葉幹細胞,確認間葉幹細胞的表面標誌,並在體外培 養刺激其分化成骨細胞(osteocytes),軟骨細胞(chondrocytes) 與脂肪細胞 (adipocytes),此外利用在試管內測試間葉幹細胞的免疫抑制功能,更進一步利用 牛第二型膠原蛋白建立類風濕性關節炎的動物模式,在疾病進程中,以靜脈注射 方式給予間葉幹細胞,用以研究其治療效果。
實驗結果:
結果顯示,脾臟中的 T 淋巴球在 anti-CD 3/28 抗體刺激下,與間葉幹細胞共 同培養,間葉幹細胞能有效抑制 T 淋巴球分裂增生,確立其免疫調控的功能。給 予間葉幹細胞可有效改善類風濕性關節炎動物模式中的發炎反應,降低疾病嚴重 程度與細胞浸潤現象 ; 血清中,經過間葉幹細胞治療的小鼠體內所產生的介白素- 1β 濃度較低 ; 膝關節萃取出的蛋白質中,介白素-6 有顯著意義地降低 ; 經間葉幹 細胞治療的小鼠脾臟細胞,可產生較高干擾素-γ(interferon-γ)介白素-6 (interleukin-6, IL-6), 介白素-10 (interleukin-10, IL-10)。
結論:
本篇實驗結果顯示,間葉幹細胞在體外培養或動物實驗中都可有效調節免疫 反應,我們認為間葉幹細胞在類風濕性關節炎的治療中有減緩的效果,未來將進 一步研究其參與在此疾病的免疫調控機轉。; Background:
Rheumatoid arthritis (RA) is a common autoimmune disease in the world. A variety of immune cells are involved in pathogenic mechanism and progression of the disease. In the early stage of RA, T lymphocytes are regarded as one of crucial cells in the disease pathogenesis and are related to disease severity. Mesenchymal stem cells (MSCs) are pluripotent stem cells obtained from many tissues. MSCs can self-renew and differentiate into several lineages under stimulations. Recently, more and more studies have demonstrated that MSCs exert the immunoregulatory capacity. In this study, we aimed to examine whether MSCs can have potential therapeutic effect on collagen-induced arthritis (CIA) in rheumatoid arthritis murine model.
Methods:
We isolated MSCs from bone marrow of DBA/1J mice. MSCs were expanded, characterized by phenotypic analysis, and differentiated into osteocytes, chondrocytes, and adipocytes. Proliferation of T lymphocytes in the absence or presence of MSCs was performed to determine the immunosuppressive function of MSCs in vitro. To investigate the effect of MSCs on CIA animal model, we immunized mice with bovine type II collagen, and injected MSCs intravenously.
Results:
MSCs efficiently inhibited the anti-CD3/28-induced T cell proliferation and division. These data suggested that MSCs had the regulatory effect on inflammatory immune responses. MSCs administration can reduce the severity of arthritis and cell infiltration. The level of IL -1β was lower in the sera of MSC-treated mice than non MSC-treated group. IL-6 expression in knee protein extraction was decreased after mice receiving MSCs. Furthermore, the lymphocytes of MSC-treated mice secreted higher IFN-γ, IL-6, and IL-10.
Conclusions:
Our results demonstrated that MSCs could play a modulatory role in the immune responses both in vitro and in vivo. The delivery of MSCs in CIA animal model ameliorated the severity of arthritis. MSCs treatment can be used as a potential therapy for RA. However, further studies are required to clarify the immunoregulatory mechanisms mediated by MSCs treatment.2012-01-01T00:00:00Z