毒理轉錄體學分析揭示環境致癌物對小鼠肺前趨細胞和人類支氣管上皮細胞株的致癌和病毒感染潛在機制

Toxico-transcriptomic analysis reveals potential mechanisms of environmental carcinogens on tumorigenesis and viral infection in mice lung progenitor cells and human bronchial epithelial cells (BEAS2B)

郭庭亦(Ting-Yi Kuo)

指導教授 ： 陳惠文

國立臺灣大學/醫學院/毒理學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202001957

摘要

隨著全球都市的高度發展，各種形式的環境污染物也隨之累積於我們的日常生活當中，經過日積月累的交互作用影響人類的健康福祉。至今為止已有許多文獻指出，惡劣的環境及空氣品質將導致更高的呼吸道及肺部病變發生率，不論是外來毒性物質或是內源性基因複製，皆會導致DNA損傷，進而增加病原體感染、發炎、致突變與罹癌的風險。此外，敏感族群，如：新生兒及老年人，有著更高的罹病風險，因此本研究使用新生鼠肺作為初級培養之細胞來源，以及人類呼吸系統細胞株來進行相關實驗，並且主要聚焦在環境常見致癌物 (如：3-Nitrobenzanthrone, 1,8-Dinitropyrene, N-Nitrosopyrrolidine)、2,3,7,8-Tetrachlorodibenzodioxin (TCDD) 與Fe2O3/static magnetic field (SMF) 的交互作用，由不同的面向來探討這些物質對肺部細胞造成的影響。
根據林泰元老師實驗室先前之研究，新生小鼠肺部有一群表現特異性細胞標誌 — Coxsackievirus/Adenovirus receptor的幹/先驅細胞族群，分離後將其命名為mPSCsCAR+，經過7~10天的培養，此標誌會逐漸消逝並且分化成類第一型肺泡上皮細胞。我們將此細胞族群與分離前的肺臟細胞 (lung primary culture cell) 以及人類呼吸系統細胞株 (BEAS2B與HOP62) 加入上述之環境污染物後以MTT assay/CCK-8觀察proliferation/migration的能力，利用免疫螢光染色 (Immunofluorescence staining) 細胞之形態變化、proliferation/migration能力與DNA 損傷的情形，再以流式細胞儀來觀察細胞週期、Real-time PCR觀察各藥物單獨或合併加入後，在特定基因上之調控。結果顯示，各環境污染物會造成不同程度的DNA 損傷，其中以1,8-Dinitropyrene最為顯著，而另一方面， 3-Nitrobenzanthrone則是會改變細胞週期 (cell cycle)使細胞停留在S-phase。此外，我們觀察到在經過NPYR刺激後，肺初級培養之細胞不論是基因還是蛋白質表現，ACE2 (Angiotensin-converting enzyme 2) 都有顯著上升，因此進而深入研究，發現mPSCsCAR+ 相較於mPSCsCAR- 此現象更為顯著，並且在加藥第七天達最高峰，之後會隨著細胞幹性降低、細胞分化而逐漸消失。由於ACE2可以做為冠狀病毒S蛋白之受體 (如：SARS-CoV-1及SARS-CoV-2)，因此推測環境污染有可能由此導致更高的病毒感染風險，然而此推論仍有被待後續實驗釐清。除此之外，我們利用DNA微陣列晶片(DNA Microarray) 去觀察不同藥物在單一或多重刺激下，大範圍基因表現在各組間的差異。最後，再探討環境污染物所改變的特定基因之表現，是否會因維他命C或白藜蘆醇，這些具有抗氧化保護力的分子的加入，而呈現出不一樣的結果。簡而言之，本篇旨在多層面地探討環境污染物對肺部細胞所造成的影響。

關鍵字

肺臟幹/先驅細胞 ； 環境致癌物 ； DNA 損傷 ； ACE2 ； DNA 微陣列

並列摘要

With the rapid development of the global metropolitan area, various forms of environmental contaminants have accumulated in our daily life through air, water, food and our living environment. These cumulative interactions could be hazard to our health and well-being. So far, many published articles have pointed out that the harsh environment and air quality can lead to a higher incidence of respiratory and pulmonary diseases. Both exogenous toxic substances and endogenous gene replication stress can cause DNA damage and increase the risks of pathogen infection, inflammation, mutagenesis and carcinogenesis. Sensitive groups, such as newborn babies and the elderly, have a far higher risk of being influenced. Therefore, this study chose the neonatal mice as the source of primary culture cells and also chose human respiratory system cell lines to conduct the relevant experiments. We mainly focused on investigating the influences of the selected carcinogens, which commonly exist in our environment, e.g. 3-Nitrobenzanthrone (3-NBA), 1,8-Dinitropyrene (1,8-DNP), N-Nitrosopyrrolidine (NPYR). And also, we dedicated into studying the consequences while these compounds encountering 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) and Fe2O3/static magnetic field (SMF) from various aspects.
Previously, Dr. Ling TY has identified a progenitor population which expressing a specific cell marker – Coxsackievirus/Adenovirus receptor (CAR) in newborn mice lung primary culture cells (mPSCsCAR+), which show the potential to differentiate into type 1 alveolar epithelial-like cells (AT1) after sorting and removal the niche-stromal cells. Here, the mPSCsCAR+ cells with or without the stromal cells were applied on studying the impacts of the environmental carcinogens; whereas, the human bronchial epithelial cell line (BEAS2B) and lung adenocarcinoma cell line (HOP62) were used for compares on cell proliferation/survival by MTT/CCK8 assays, cell morphological changes and DNA damage through immunofluorescence staining, cell cycle analysis via flow cytometry, and real-time Q-PCR to detect the APOBEC- and carcinogen metabolism-related gene expression under various treatments individually or in combination. The results show that these environmental pollutants can cause different levels of DNA damage, of which 1,8-DNP is the most significant. Besides, 3-NBA could promote cell cycle progression with significantly increase the ratio of S-phase. Interestingly, we observed that NPYT could up-regulate angiotensin-converting enzyme 2 (ACE2) gene expression or protein level in lung primary culture cells, and confirmed that this finding is mainly attributed by mPSCsCAR+ population. The fold change of ACE2 gene expression will reach its peak and gradually decrease as the drop of the cell stemness characteristic accompanied by cell differentiation. Since ACE2 act as a cellular entry receptor for coronavirus (eg: SARS-CoV-1 and SARS-CoV-2) and expression levels of ACE2 might positive correlate to efficiency of virus entry, it is suggested that environmental pollution may results in a higher hazard of viral infection. The subsequent experiments to support the finding is urgently needed. Moreover, we utilized transcriptomic analysis to observe the toxico-genetic regulation in the large-scale gene expression map under single or multiple toxicants stimulation. We found that certain encoded proteins possess the similar functions would be up- or down-regulated under different treatments simultaneously which may relate to some evolving pathways in mice, such as immunity, development and metabolism. Lastly, we examined that if the addition of vitamin C or resveratrol, the molecules that possess antioxidant property, could show the benefit or worse to the toxicant effects. In conclusion, this thesis might add our understanding on realizing the influence of environmental pollutants in the pathogenesis of the lung diseases, e.g., lung cancer or virus infection.

並列關鍵字

lung stem/progenitor cell ； environmental carcinogens ； DNA damage ； lung cancer ； microarray

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