- 學位論文
祕魯苦蘵成分AN37選擇性殺死口腔癌細胞株之轉錄體圖譜
Transcriptome profiles for selective killing of oral cancer cells by Physalis peruviana-derived compound AN37
摘要
AN37是單一化學結構的純化物，純化於秘魯苦蘵(Physalis peruviana)植物全株。在實驗室先前的研究中發現此純化物對口腔癌細胞有選擇性殺傷之效果。在彗星實驗結果中，發現AN37可以促使口腔癌細胞株Ca9-22受到DNA損傷更勝人類正常牙齦纖維細胞株(human gingiva fibroblast; HGF-1)。在細胞週期分析的結果中，發現相較於HGF-1細胞，AN37可以促使口腔癌細胞株Ca9-22產生更多比例的Sub-G1時期。但AN37如何對口腔癌細胞有選擇性殺傷效果之細胞機制仍然不明，故本研究重點在於利用系統生物學分析其轉錄體圖譜與其相關機制與路徑。本實驗中，將HGF-1與Ca9-22細胞株分別加入相同劑量，即Ca9-22細胞株的AN37 IC50劑量3.14 ?慊/ml後，處理不同時間間隔：2、8和24小時，再利用生物晶片技術來測定mRNA變化量，並使用生物資訊軟體(MetaCore™) 進一步分析。AN37處理後的 Ca9-22和HGF-1細胞分析數據進行比較後，發現選擇性殺傷途徑有許多可能性。在這些不同的途徑中，本研究在分析所有途徑後，目前只選擇DNA damage pathway map:「ATM and ATR regulation of G1/S checkpoint」，做進一步進行即時定量聚合酶連鎖反應去驗證候選基因。最後，篩選出多個具有差異性的十八個基因 (ATRIP, BLM, BRCA1, CCNA1, CCND1, CCNE1, CDC25A, CDK4, CDKN1A, CHEK1, CHEK2, CLSPN, MDM2, NBN, NFBD1, NFKB1, NFKBIE與USP1)做為目標基因，以利未來藉此找出使AN37具有選擇性殺傷能力的關鍵候選基因。
並列摘要
AN37 is a pure compound derived from Physalis peruviana. In our previous study, AN37 displayed the selective killing ability in oral cancer cell. We found that treatment with AN37 causes more DNA damage in oral cancer cell (Ca9-22) than normal cell (human normal gingiva fibroblast, HGF-1). Cell cycle analysis indicates that AN37 can promote Ca9-22 to undergo Sub-G1 arrest than HGF-1. However, the detailed mechanism insight into selective killing of AN37 against oral cancer is still unknown. In this thesis, the systems biology strategy based on microarray study, bioinformatics, and real-time PCR were applied to investigate the selective killing mechanism of AN37. Ca9-22 and HGF-1 cells were treated with IC50 dose of AN37 (3.14 ?慊/ml) for 2, 8 and 24 h for microarray experiments. Using bioinformatics data mining tool- MetaCore™, networks and candidates with differential expressions between oral cancer and normal cells were screened. I choose DNA damage pathway map: “ATM and ATR regulation of G1/S checkpoint” and validation by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). Finally, we identified 18 genes by the certification (ATRIP, BLM, BRCA1, CCNA1, CCND1, CCNE1, CDC25A, CDK4, CDKN1A, CHEK1, CHEK2, CLSPN, MDM2, NBN, NFBD1, NFKB1, NFKBIE, and USP1) providing selective killing gene candidates which warrants further investigation in the oral cancer therapy.