余幸司臺灣大學：臨床醫學研究所何鈞軒Ho, Chun-HsuanChun-HsuanHo2007-11-272018-07-062007-11-272018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/55449台灣西南部沿海高度砷污染之地區常有區域性砷癌發生(包括: 皮膚癌、膀胱癌、腎臟癌、肺癌、大腸癌、及肝癌等)。在皮膚組織中，表皮角質細胞為砷的標靶細胞。慢性砷中毒症的皮膚表現則包括色素沉著、角化及癌症。砷引起的皮膚癌又以波文氏病 (Bowen’s disease, carcinoma in situ) 最為常見，為一具特異性的本土性環境病。砷引起的波文氏病之病理特徵有 (1)表皮細胞增生(2)表皮細胞排列失去極性以及惡性化(3)波文氏病中有大量之發炎細胞浸潤(4)病變表皮內見散在性凋亡細胞。然而，我們卻發現砷引起波文氏病組織中浸潤之淋巴球會有特殊的凋亡現象，但其真正致病機轉，以及免疫細胞和角質細胞之間的交互作用，目前並不是非常清楚，仍需要進一步的研究，也是本實驗的主題。過去，砷對角質細胞的作用機轉已有相當多的研究。現在，我們已知— 1.在低濃度下（0.1∼1μM），砷透過NF-κB調控G1期之cyclinD1/CDK4、S期cyclin A/CDK2及G2期之cyclinB1/CDK1等細胞週期調控蛋白表現，造成角質細胞增生。 2.在高濃度下（> 1μM），則會經由AP1活化Fas/Fas ligands以及抑制NF-κB，導致角質細胞程序凋亡。 3.砷可促進巨噬細胞釋出TNF-α，並經由TNF receptor-1 的調控引起末稍血液CD4+ T 細胞凋亡。 4. 砷處理後的角質細胞經由活化Fas/Fas ligands，造成CD4+ T 細胞凋亡然而，有趣的是，根據臨床實驗證實，長期暴露於砷污染環境的病人，其血液中的CD4+ T 細胞，經過適應，反而對砷所造成的細胞毒性產生抵抗性。因此，我們可以推測：在砷引起波文氏病組織中浸潤的淋巴球會產生凋亡現象，其並非直接來自砷本身的毒性，而是透過其他的作用機轉。根據以上種種證據，我們假設，在砷作用下，皮膚組織會引起慢性發炎，在這樣的環境下，角質細胞會透過分泌Fas/Fas ligands，逆向造成淋巴球細胞的凋亡，進而避免免疫細胞的攻擊，進而轉型成為皮膚癌。為證實這樣的想法，本實驗分為三大部分： 1. In vivo：透過砷引起波文氏病組織的CD4、CD8、TUNEL 和Fas ligand 的特殊染色比對發炎組織中凋零的細胞是否為CD4+ T 細胞，另外，也可證實角質細胞表面Fas ligand 的量是否增加？ 2. In vitro：A.在淋巴球中加入soluble Fas ligand，觀察是否會引起凋亡B.在角質細胞中加入砷，是否會促使釋放soluble Fas ligand？ C.取砷處理後之培養角質細胞的上清液，加入淋巴球中，觀察是否 有凋亡的情形？ 3. In organotypic culture：以擬態皮膚培養模組，透過A.表皮層加砷B.真皮層加砷C.對照組三種不同的傳送方式，來模擬砷引起波文氏病組織的致病過程，透過生化分析、特殊染色、laser captured microdissection 以及microarray…等技術，進而和砷引起波文氏病組織以及砷處理後的培養細胞作為比對，觀察角質細胞和淋巴球細胞之變化，來驗證實驗的假設。 從我們的實驗結果證明，砷引起的波文氏症細胞透過分泌Fas/Fas ligands，造成淋巴球的凋亡，進而抑制免疫細胞的攻擊，此即為所謂免疫特免（immune privilege），這樣的情況在其他皮膚癌組織中，也已經被觀察到。最後，希望此實驗能提供一種用以探討癌化角質細胞之免疫防衛機轉的研究平台。Endemic arsenic-induced cancers, ex: skin cancer, bladder cancer, kidney cancer, lung cancer, colon cancer and liver cancer…etc, develop frequently over the highly arsenic-polluted areas in the southwestern Taiwan. In skin tissue, keratinocytes are the target cell of arsenic intoxicity. The dermatologic presentations of chronic arsenism include hyper-pigmentation, hyper-keratinization and malignancy. Among arsenic-induced skin cancer, bowen’s disease, a kind of carcinoma in situ, is the most common and an endemic environmental disease with specificity. The pathological features of arsenic-induced bowen’s disease are: (1) epidermal proliferation (2) loss of cell polarity and atypical keratinocytes (3) massive lymphocytic infiltration in the dermis (4) intra-lesional apoptotic keratinocytes. However, we discovered the characteristic apoptosis of lymphocytes infiltrated in the arsenic-induced bowen’s disease, that never mentioned in the documents before. Both of the underlying pathogenesis and the interaction between keratinocyte and lymphocyte are still unknown and need further investigation. Those are also the goals of this study. In the past, the effects of arsenic to keratinocytes had been well-studied. Now, we have known – 1. At low concentration（0.1∼1μM）, arsenic could induce the proliferation of keratinocytes via control the expression of cell-cycle regulatory proteins, ex:cyclinD1/CDK4 of G1 phase, cyclinA/CDK2 of S phase and cyclinB1/CDK1 of G2 phase via NF-κB. 2. At high concentration（> 1μM）, arsenic turn to activate Fas/Fas ligands by AP-1 and inhibit NF-κB, that could lead into the apoptosis of keratinocytes. 3. Arsenic could promote the macrophages to release TNF-α and induced the apoptosis of CD4+ T cell in the peripheral blood via the regulation of TNF receptor-1 4. Arsenic-treated keratinocytes induced the apoptosis of CD4+ T cell via activation of Fas/Fas ligands On the contrary, according to the clinical data, the CD4+ T cell in the peripheral blood of the patients with chronic arsenism is more resistant to the arsenic cytotoxicity after long-term adaptation. Hence, we speculated that the apoptosis of lymphocytes infiltrated in the arsenic-induced bowen’s disease is not directly caused by the toxicity of arsenic, but the other mechanism. So the hypothesis of this study is that the cancer cell of bowen’s disease could induce the apoptosis of CD4+ T cell via activation of Fas/Fas ligands. We will confirm this assumption through three parts of study designs, including 1. in vivo 2. in vitro 3. in organotypic culture. Our goal is to clarify the molecular mechanism of apoptotic lymphocytes infiltrated in arsenic-induced Bowen’s disease and the role of immune defense in skin cancer.一、中文摘要…………………………………………………………3 二、緒論……………………………………………………………….4 三、研究方法與材料…………………………………………………10 四、結果 第一部份………………………………………………………..15 第二部份………………………………………………………..15 第三部分………………………………………………………..16 五、討論……………………………………………………………....17 六、展望……………………………………………………………....20 七、論文英文簡述…………………………………………………...21 八、參考文獻………………………..………………………………..22 九、圖表………………………………………………………………27 十、附錄………………………………………………………………46811321 bytesapplication/pdfen-US淋巴球砷波文氏病凋亡arseniclymphocyteapoptosis[SDGs]SDG3texthttp://ntur.lib.ntu.edu.tw/bitstream/246246/55449/1/ntu-95-P93421014-1.pdf