- 學位論文
砷致人類角質細胞及淋巴球細胞凋亡分子調控機制之研究
Study of Molecular Mechanisms in Arsenic-induced Apoptosis in Human Keratinocytes and Lymphocytes
摘要
砷可於多種組織引發癌症。於皮膚組織，砷暴露可引起多發性之表皮內癌波文氏病 (Bowen’s disease)，是砷引起的代表性疾病之一。波文氏病組織切片有角質細胞癌化、增生及細胞凋亡 (Apoptosis) 的情形。此外，波文氏病患者有免疫功能低下的情形，其血液淋巴球中的輔助型T細胞數目減少，並有細胞功能異常的現象。本論文基於這些砷引發之波文氏病的觀察，針對在波文氏中有異常的角質細胞及淋巴球細胞以體外培養模式進行研究，以探討砷致人類角質細胞及淋巴球細胞異常的作用機轉。研究由砷對人類角質細胞的影響及砷對人類淋巴球細胞的影響等二部分的分析組成，最後再將此二部分的結果與臨床的觀察加以對照，以評估我們在體外培養實驗所得之分子作用機轉是否適用於解釋人體的反應。 研究結果顯示，在角質細胞，濃度1 micro M 的砷會引起NF-kB為主的訊息調控異常，促進包括G1期細胞週期之cyclin D1/CDK4，S期之cyclin A/CDK2及M期之cyclin B1/CDK1等細胞週期調控蛋白的表現，最後造成角質細胞增生。濃度5-10 micro M 的砷則會抑制NF-kappa B，活化AP-1，並引發以P53為主的細胞週期抑制，包括G1期及M期之細胞週期調控蛋白表現量降低，及細胞週期抑制蛋白p21 waf1/cip1 及14-3-3 sigma表現量增加，造成G1及M細胞週期之停滯，並會引發由Fas/FasL調控的細胞程序凋亡。我們證實砷隨著濃度的不同，對人類角質細胞具有雙向性的影響。在人纇淋巴球方面，砷則沒有如同對人類角質細胞的雙向性影響。濃度1 mM或以上的砷會促進培養的人纇淋巴球細胞釋出TNF-alpha，並會造成細胞程序凋亡。進一步分析凋亡的細胞族群，顯示淋巴球中CD4+的輔助型T細胞是主要的細胞凋亡族群。我們發現砷會促進輔助型T細胞上TNF receptor 1 的表現，並經由TNF-alpha與TNF receptor 1的訊息調控，特異性的引發人類淋巴球中的輔助型T細胞程序凋亡。本論文研究結果中砷致角質細胞增生/凋亡的情形，與臨床觀察之波文氏病相似。此外，砷引起輔助型T細胞程序凋亡的特性，與在波文氏病患血液輔助型T細胞減少的情形有一致性，可能與波文氏病患者免疫功能低下有關，因此我們進一步將波文氏病組織以CD4及TUNEL 染色，結果發現在波文氏病組織的浸潤淋巴球中，有特異性的輔助型T細胞凋亡的情況，證實了我們在體外培養實驗所得的結果，確實在臨床患者體內發生，可作為砷之生物效應的參考。 本研究以砷致表皮角質細胞癌化具代表性的波文氏病徵為出發點，分別對砷在病變的角質細胞本身及病變相關的免疫細胞中的作用機轉加以探討，所得結果皆與臨床觀察之波文氏病皮膚砷癌相似，期望這些成果能對於瞭解砷在波文氏病致病機轉中所扮演之角色有所助益。
並列摘要
Exposure to arsenic induces arsenical cancers in multiple tissues. In the skin, Bowen’s disease is the most significant arsenic-related tumor. Immunohistochemically, Bowen’s disease shows carcinogenic, proliferating and apoptotic characteristics. Furthermore, Bowen’s disease shows defective immune functions, including the dysfunction and decreased T helper cells in peripheral lymphocytes. To elucidate the molecular mechanisms in arsenic-induced Bowen’s disease, this study investigated the arsenical effects on cultured human keratinocytes and lymphocytes. The results from in vitro models were compared to the clinical observations in Bowen’s disease to demonstrate possible mechanisms to explain how arsenic may act in vivo. Our results indicated that 1 micro M of arsenic enhanced NF-kappa B activity, which was related to increased expression of cell cycle regulatory proteins (including cyclin D1/CDK4, cyclin A/ CDK2 and cyclin B1/CDK1) and resulted in keratinocyte proliferation. At higher concentrations (5-10 micro M), arsenic suppressed NF-kappa B activity, enhanced AP-1 activity, and triggered the p53-related cell cycle arrest. Decreased levels of G1 and M phase-specific cell cycle regulatory proteins were associated with increased levels of cell cycle inhibitory proteins, such as p21 waf1/cip1 and 14-3-3 sigma. At these arsenic concentrations, keratinocyte cell cycle was arrested at G1 and M phase. In addition, apoptosis was induced via Fas/FasL pathway in keratinocytes. These results demonstrated the biphasic effects of arsenic on keratinocytes. However, these biphasic effects by arsenic were not observed in normal lymphocytes. Arsenic concentrations higher than 1 micro M induced TNF-alpha release from human lymphocytes and caused a apoptotic effect. CD4+ cells (T helper cells) were the major apoptoic population in human lymphocytes. TNF receptor-1 (TNF-R1) expression on T helper cells was significantly enhanced by arsenic treatment when compared to other cell populations in lymphocytes. We found that arsenic specifically induced T helper cell apoptosis via TNF receptor-1 pathway. The in vitro results of arsenic-induced keratinocyte proliferation and apoptosis, as well as arsenic-induced T helper cells apoptosis, are similar to the clinical observations in Bowen’s disease. These results imply that arsenic-induced T helper cell death is correlated to defects in immune function. Therefore, we futher stained the apoptotic patterns of infiltration lymphocytes in Bowen’s disease lesion. And the T helper cell-specific apoptosis was observed in the infiltration lymphocytes. These results indicated that our in vitro findings are able to explain the pathological events in Bowen’s disease. This study investigates the effects of arsenic on human keratinocytes and lymphocytes based on the pathological characteristics in arsenic-induced Bowen’s disease. We hope our findings will be helpful for better understand the molecular mechanisms in arsenic-induced Bowen’s disease.
參考文獻
延伸閱讀
- TU, P. C. (2005). 細胞凋亡相關蛋白表現與光動力治療效果關係之研究 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2005.00844
- 卓映君（2010）。凋亡細胞清除相關基因多型性與台灣地區全身性紅斑性狼瘡及類風濕性關節炎發生之探討〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2010.00360
- 呂偉台（2022）。探討 1-Undecanol 誘導人類角膜上皮細胞之氧化壓力、細胞焦亡及分子機轉〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/csmu202200153
- 洪苓方（2001）。Studies on the Molecular Mechanisms of Ketoconazole-induced Apoptosis in Human Prostate Cancer Cell Lines〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-1704200714264628
- 黃忠彥（2012）。Study on molecular mechanism of Lipocalin-2 induces apoptosis in human hepatocellular carcinoma cells〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2012.00108