Cellular Responses to Benzo(a)pyrene in Clara Related Stem/Progenitor Cells

苯芘benzo(a)pyrene (BaP)，為多環芳香烴物質(Ploycyclic Aromatic Hydrocarban, PAHs)，主要大量存在於香菸煙狀物、汽機車排放物及食物燒烤物。BaP 於細胞中經過細胞色素P450及epoxide hydrolase代謝活化生成中間產物BaP diolepoxide (BPDE)，BPDE攻擊DNA及大分子蛋白生成供價鍵結，而被認為是BaP致突變性的主要途徑。BaP 已被証實於實驗動物為致突變性、致腫瘤及對人體為可能性致癌，國際癌症中心(International Arctic Research Center, IARC)將BaP列為可能的人類致癌物質(group 2A)之等級。而肺臟是呼吸系統面對外來物的第一防線，其中Clara cells位於支氣管束、支氣管與肺泡交接處被視為是肺臟幹源細胞，含豐富細胞色素-450對外來物之代謝行為具重要角色，但也因此成為許多毒物之目標細胞。文獻報導指出當BaP進入生物體後，在肺部Clara cells及Alveolar type II有高含量累積，引發本篇研究目的在於探討當Clara cells在面對BaP這樣一個經代謝後具致癌性的毒物時細胞之相關反應為何。驗利用林泰元博士於2006年發表於美國國家科學院院刊，取老鼠肺臟以無血清方式進行原代培養，建立原代培養細胞中包括Clara related stem/progenitor cells以及mesenchymal stroma cells。實驗首先利用免疫細胞染色以及反轉錄聚合酶鏈鎖反應鑑定細胞表現octamer-binding transcription (Oct4)及Clara cell secretion protein (CCSP)蛋白，確定細胞型態與成熟狀態。接著以0.5至50 μM BaP處理原代培養細胞，24小時後發現細胞增生形情。而分析細胞週期分佈發現，BaP的處理造成S期上升。進一步以BrdU以及CCSP染色標記S期細胞和Clara related stem/progenitor cells發現BaP造成的細胞週期S期上升，是發生在Clara related stem/progenitor cells，然而對mesenchymal stroma cells的細胞週期則沒有影響。aP是aryl hydrocarbon receptor(AhR)的ligand，當它進入細胞後會與細胞質的AhR結合發生核轉移，進入細胞再與ARNT活化下游基因表現造成細胞增生以及對外來物的代謝活性。實驗利用反轉錄聚合酶鏈鎖反應以及免疫細胞染色發現於BaP處理4小時後發生AhR核轉移，而活化代謝酵素CYP1A1，CYP1B1表現於12，24小時上升2倍。接著利用即時反轉錄聚合酶鏈鎖反應定量發現，c-myc表現量隨BaP處理時間而上升，於24小時增加至3.5倍。c-myc下游分子的影響，包括了cyclin D1表現於BaP處理24小時增加至4.2倍，CDK4蛋白的表現增加至3.5倍。由此推測BaP造成的S期上升是來自c-myc的活化，因此實驗進一步利用siRNA抑制c-myc作用，發現c-myc的抑制有效地減少BaP誘導之S期上升。BaP經代謝活化後生成活性中間產物BPDE是致突變性的來源。實驗利用免疫細胞染色可觀察到BPDE-DNA鍵結物的生成，而BaP於原代培養細胞是否造成基因毒性，利用慧星試驗發現BaP處理24小時後與基因毒性的現象相較於陰性對照組具統計意義增加。目前實驗發現，BaP的處理造成c-myc活化，進而啟動下游分子造成細胞週期進入S期。同時BaP的作用於原代培養細胞中造成基因毒性。綜合以上結果推測: BaP處理原代培養細胞於Clara related stem/progenitor cell造成細胞週期進入S期可能與c-myc及其下游分子相關，同時伴隨著BaP所造成的基因毒性細胞可能啟動DNA修補機制而使細胞週期有S期停滯情形，實驗未來將針對基因毒性啟動之DNA修補機制進一步探討。最後，BaP是已知的可能人類致癌物質，Clara related stem/progenitor cells 在進行代謝同時，所生成的BPDE造成之基因毒性是否誘導細胞走向癌化則是本實驗未來方向。Benzo(a)pyrene (BaP) belongs to polycyclic aromatic hydrocarbon (PAH) family of environmental carcinogens that are present in tobacco smoke , barbecued food and auto exhaust. BaP require cytochrome P450 and epoxide hydrolase mediated bio-activation for generation of their respective ultimate carcinogenic dio epoxide intermediates, (+)benzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE). Covalent interation of BPDE with DNA is believed to be a critical step in BaP-induced tumorigenesis. BaP have been implicated in the etiology of lung cancer. In the lung, BaP was located in ciliated and non-ciliated Clara and alveolar type II cells, resulting in the experimental animals, such as rat, mice, or rabbit. Clara cells play important role in the metabolism of xenobiotic in the lung, because they have abundant cytochrome P450 monooxygenase. In previously studies, Clara related stem/progenitor cells were identified and generated in lung stem/progenitor cell. Although, the potential roles of Clara cells in metabolism of xenobiotic have been proposed, however, the molecular mechanism of BaP metabolically activation in the Clara related stem/progenitor cells still unclear. n this work, we set a lung stem/progenitor cell including Clara cell related stem/progenitor cells surrounding with mesenchymal stroma cells to examine the BaP-induced cell response. Different concentration of BaP treatments were applied to lung stem/progenitor cells. The MTT assay showed the high concentration of BaP cause cell proliferation in lung stem/progenitor cells. The cell cycle analysis revealed that S phase increased after BaP treatment for 24 hours. By BrdU incorporation experiment showed that BaP exposure was able to promote Clara related stem/progenitor cell entry to S phase.BaP is a ligand of aryl hydrocarbon receptor(AhR), binding leads to nuclear translocation and interaction with its partner protein AhR nuclear translocator (ARNT) will active xenobiotic metabolism and proliferation. By mRNA and immunocytochemistry analysis, BaP exposure caused AhR translocation to nuclei at 4 hours and activated CYP1A1, CYP1B1 expression at 12 and 24 hours. Further, BaP-induced c-myc, cyclin D1 expression up to 3.5 and 4.2 folds by real-time PCR and CDK4 protein level up to 3.5 folds by western blot analysis. Knockdown of c-myc in lung stem/progenitor cells by siRNA transfection, suppressed S phase distribution for BaP treatment.BaP can be metabolized into BPDE, a ultimate of carcinogen attach to DNA cause genotoxicity. From immunocytochemistry of BPDE DNA positive result reveal that BaP probably cause BPDE DNA adduct in Clara related stem/progenitor cells. And using comet assay, BaP has been shown to cause genotoxicity. he current study showed that BaP activates c-myc expression of lung stem/progenitor cell, and through cyclin D1/CDK4 mediated cell cycle entry to S phase. Meanwhile, BaP treatment also induced CYP1A1 expression and genotoxicity in Clara related stem/progenitor cells. Together, these results indicated that BaP presented the potent carcinogen for lung stem/progenitor cells to undergo tumorigenesis processes. And hinted that S phase increased caused from cell cycle progession by BaP-induced c-myc activation and might result in arrested by genotoxity induced repair mechanism.圖表目錄---------------------------------------------- II文摘要---------------------------------------------- III文摘要---------------------------------------------- V寫表------------------------------------------------ VII一章 緒論---------------------------------------- 1二章 實驗材料與方法-------------------------------- 10三章 實驗結果-------------------------------------- 29四章 實驗討論-------------------------------------- 35五章 參考文獻-------------------------------------- 43六章 圖表集---------------------------------------- 4