非小細胞肺癌（NSCLC）為國人癌症死亡主因，表皮生長因子受體（EGFR）的異常表現是NSCLC惡化的關鍵途徑之一。EGFR-酪氨酸酶抑制劑（TKIs）為EGFR突變之NSCLC治療用藥，然而多數患者常在治療後對TKIs產生抗藥性。目前已知PM 2.5 （Particulate Matter 2.5）與肺癌發展相關，然而PM 2.5促使NSCLC惡化和產生抗藥性等影響仍無定論。本研究探討PM 2.5是否藉由活化EGFR途徑促使肺癌快速惡化以及是否干擾TKIs治療效果。本實驗利用各種EGFR狀態之細胞株平台長期3個月給予PM 2.5和/或TKI，以模擬肺癌患者在治療期間暴露於PM 2.5，此模式也運用轉錄體學分析分子作用機制。目前實驗結果指出：NSCLC細胞暴露於PM 2.5會促使EGFR途徑活化、干擾TKIs治療效果、誘導DNA損傷、並會促進細胞生長和爬行。使用轉錄體學分析指出長期暴露於PM 2.5下，跨膜絲胺酸蛋白酶2（TMPRSS2）表現異常增加，目前已知TMPRSS2與前列腺癌發展和促使SARS-CoV-2感染肺細胞有關，未來進一步釐清PM 2.5對NSCLC細胞中TMPRSS2的調控機制，將有助了解PM 2.5是否與肺癌長年居國人第一奪命癌的關聯，更有助於發展治療新策略。
關鍵詞：PM 2.5、非小細胞肺癌、表皮生長因子受體、DNA損傷、跨膜絲胺酸蛋白酶2

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in Taiwan. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are approved treatment for NSCLC patients harboring EGFR activating mutations. PM 2.5 (Particulate Matter 2.5) is related to the incidence and mortality rate of lung cancer and less is known concerning the detailed machines through which PM 2.5 exposure promotes lung cancer progression or interference with TKIs treatment. In this study, we performed short (24 hours) and long term (3 months) exposure models to investigate the pathogenic effects of PM 2.5 in NSCLC cells harboring various EGFR statuses. Our results indicate that both in short and long term PM 2.5 exposure upregulated EGFR signaling pathways and interfered TKIs sensitivity in NSCLC cells. Long-term PM 2.5 exposure further enhanced proliferation, anchorage-independent growth, and induced DNA damage in NSCLC harboring EGFR mutation cells both in vitro and in vivo. Transcriptome analyses reveled that the level of the transmembrane protease serine 2 (TMPRSS2) was increased in PM 2.5-long term treated cells. Furthermore, it may be worth investigating the involvement of the mechanisms by which PM 2.5 increases the expression of TMPRSS2 in NSCLC cells. Taken together, our findings provide experimental evidence that exposure to PM 2.5 may adversely affect the progression and TKIs sensitivity for the pathological features of NSCLC.
Keywords：PM 2.5, NSCLC, EGFR, DNA damage, TMPRSS2

目 錄
中文摘要 iv
英文摘要 v
目錄 vi
圖目錄 ix
表目錄 xii
第一章 緒論 1
第一節 肺癌（Lung cancer） 1
第二節 表皮生長因子受體（Epidermal growth factor receptor， EGFR） 2
第三節 PM 2.5 4
第四節 PM 2.5對呼吸系統的影響 7
第五節 PM 2.5對EGFR信號途徑的影響 8
第六節 PM 2.5在肺癌細胞層次的研究 9
第二章 研究目的 11
第三章 實驗材料與方法 12
第一節 試劑（Reagents） 12
第二節 細胞株及培養（Cell lines and culture）13
第三節PM 2.5之粒徑分析（Particle size determination of PM 2.5） 15
第四節 台盼藍分析（Trypan blue assay） 15
第五節 西方墨點法（Western blot） 16
第六節 抗體（Antibodies） 19
第七節 細胞爬行試驗（Wound healing assay） 20
第八節 活性氧檢測（ROS measurement） 20
第九節 反轉錄聚合酶連鎖反應（Reverse transcription-polymerase chain reaction，RT-PCR） 20
第十節 免疫螢光染色（Immunofluorescence）與雷射共軛焦顯微鏡（Laser scanning confocal microscope） 23
第十一節 軟瓊脂群落形成分析（Soft agar colony formation assay） 24
第十二節 細胞週期分析（Cell cycle analysis） 24
第十三節 細胞激素檢測套組（Cytokine ELISA kits） 25
第十四節 動物實驗（Xenograft model） 26
第十五節 統計分析（Statistical analysis） 27
第四章 實驗結果 28
第一節 探討PM 2.5對肺部正常纖維母細胞株及肺癌細胞株的惡性影響 28
第二節 探討PM 2.5對NSCLC細胞中EGFR活化途徑及TKIs作用的影響 30
第三節 探討PM 2.5長期暴露對帶有突變型EGFR之NSCLC細胞中引起的DNA損傷修復評估 32
第四節 使用轉錄體學分析長期暴露PM 2.5對肺癌細胞影響 35
第五節 以動物模式評估長期暴露於PM 2.5對NSCLC細胞的癌化影響力 37
第五章 討論 38
參考文獻 69

圖目錄
圖1. PM 2.5對肺部正常纖維母細胞株及肺癌細胞株的毒殺作用 46
圖2. PM 2.5短期24小時暴露對帶有突變型EGFR之 H1975 細胞遷移的影響 47
圖3. PM 2.5短期24小時暴露對帶有野生型EGFR之A549細胞遷移的影響 48
圖4. PM 2.5長期暴露對帶有野生型或突變型EGFR之NSCLC細胞增殖的影響 49
圖5. PM 2.5長期暴露對帶有突變型EGFR之NSCLC細胞anchorage independent growth能力的影響 50
圖6. PM 2.5短期暴露對帶有野生型或突變型EGFR之NSCLC細胞中EGFR相關途徑的影響 51
圖7. PM 2.5短期暴露對帶有野生型EGFR之國人NSCLC患者肋膜積水中分離出的肺癌細胞CL83中EGFR相關途徑的影響 52
圖8. PM 2.5長期暴露對帶有野生型EGFR之NSCLC細胞中EGFR相關途徑的影響 53
圖9. PM 2.5對帶有EGFR之NSCLC細胞中於TKIs作用後對於EGFR相關途徑的影響 54
圖10. PM 2.5對帶有突變型EGFR之NSCLC細胞中於TKIs作用後對於EGFR相關途徑的影響 55
圖11. PM 2.5對帶有突變型EGFR之國人NSCLC患者肋膜積水中分離出的肺癌細胞CL25於TKIs作用後對EGFR相關途徑的影響 56
圖12. PM 2.5長期暴露對帶有突變型EGFR之NSCLC細胞中細胞週期的影響 57
圖13. 探討PM 2.5長期暴露在帶有突變型EGFR之NSCLC細胞中引起的DNA損傷 58
圖14. 探討PM 2.5長期暴露在帶有突變型EGFR之NSCLC細胞中引起的DNA損傷 59
圖15. PM 2.5暴露下NSCLC細胞TEM分析 60
圖16. 探討PM 2.5暴露下在NSCLC細胞中ROS的產生 61
圖17. 探討PM 2.5長期暴露下在NSCLC細胞中細胞激素的產生 62
圖18. 使用轉錄體學技術尋找在PM 2.5作用後H1975細胞中有表現差異的基因 63
圖19. TMPRSS22可能在肺癌中作為致癌基因影響肺癌細胞發展 64
圖20. 動物模式評估長期3個月暴露於PM 2.5對NSCLC細胞致瘤能力 65
圖21. 透過IHC評估PM 2.5長期暴露下小鼠腫瘤組織中目標蛋白的表現 66
圖22. 空氣懸浮微粒PM 2.5對肺癌惡化發展的影響示意圖 67

表目錄
表1. TMPRSS2是PM 2.5存在之H1975細胞中基因表現有增加的排行中排名第十 68

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