臺灣博碩士論文加值系統

根據美國癌症協會之統計資料顯示，肺癌為目前世界上最常見及致命之癌症之一。現今中草藥已用於癌症治療上。於本試驗中，我們期望從中草藥當中篩選出具抑制肺癌細胞之增生及血管新生之藥物。首先，我們構築出冷光報導基因載體，以細胞轉染方式將質體送入人類肺腺癌細胞株(CL1-0)；並以藥物(G418)篩選出穩定表現冷光之CL1-0細胞株(CL1-0/luc+)。利用CL1-0/luc+之特性，以50 μM及5 μM濃度之中草藥萃取物(Chinese herb medicines, CHM)處理細胞24小時，並測其冷光報導基因活性，找出45種中草藥萃取物之細胞毒殺死亡率20% (IC20)之實驗濃度，並以個別藥物之IC20進行後續試驗。傷口癒合試驗(wound-healing assay)結果得知細胞生長受中草藥影響，45種中草藥內其中13種可抑制細胞生長能力20%以上，並以此13種中草藥進行後續試驗。於雞胚胎絨毛尿囊膜進行血管新生試驗(Chorioallantoic Membrane assay, CAM assay)，將含有中草藥之gelatin-agar放置於CAM上處理76小時後，拍照記錄血管新生情形；由結果可發現13種中草藥之中，其中12種中草藥具抑制血管新生能力；且這12種中草藥物之中，其中4種中草藥具抑制血管新生能力達約75%。於細胞遷移試驗 (transwell migration assay)測試上述4種中草藥之抑制細胞移動能力，發現其中CHM11具有顯著抑制效果達86%；另3D腫瘤侵入試驗之結果，同樣發現CHM11顯著抑制CL1-5細胞侵入能力約23%。文獻指出，HLJ1之表現量與肺癌細胞株之侵入能力呈反比；HLJ1為抑制癌轉移之基因，大量表現HLJ1基因可抑制肺癌細胞侵入之能力。我們以Real-time PCR獲得類似結果，CL1-5之HLJ1表現量因中草藥的結果而提升，遂以轉錄因子搜尋網站(TESS)分析HLJ1啟動子序列，發現HNF1及IRF1兩個轉錄因子結合位，而以電泳移動分析(Electrophoretic mobility shift assay, EMSA)結果發現，HNF1之結合力因4種中草藥之添加提升，因此推測HNF1具有結合至HLJ1啟動子區域進而提升HLJ1表現量之能力。

According to American Cancer Society statistics of 2012, lung cancer has become the most common and lethal disease in the world. Herbal medications are now used in cancer therapy. In our study, we expect to screening the health-care herbs whether can control the proliferation of lung cancer cell and angiogenesis. First, we used luciferase assay to determine IC20 of forty five traditional Chinese medicines in CL1-0/luc+ cell line. CL1-0/luc+ cell were treated with 50 μM or lower concentration of herb medicines, after 24 hours, we measured the luciferase activity by reporter gene assay. Then we performed wound-healing assay to investigate which herb medicines can suppress cancer cell proliferation. Our result shows that there are thirteen traditional herb medicines can suppress cancer cell proliferation. We further performed Chorioallantoic Membrane assay (CAM assay) with these thirteen herb medicines. By analyze the branch of vessels, we found that there are twelve herb medicines could block angiogenesis ability, and four of these herb medicines strongly reduce angiogenesis ability. In transwell migration assay and 3D spheroid invasion assay, we discovered that CHM11 can inhibit the migration and invasion ability of CL1-5. In previous reports showed that HLJ1 is a novel tumor suppressor, and we also found that HLJ1 expression increase by CHM addition. We further analyzed the promoter region of HLJ1 that we found HNF1 and IRF1 transcription factor binding site. In the results of electrophoretic mobility shift assay, HNF1 binding ability is increased by CHM1、5、11、31, hence HNF1 could be the activator of HLJ1.

致謝辭 i
中文摘要 ii
英文摘要 iii
目次 4
圖表目次 6
第一章 文獻探討 7
第一節 肺癌 7
一、 現今國人肺癌發生率與死亡率 7
二、 癌症發生原因 7
三、 癌細胞種類 7
四、 肺癌分期與治療 8
第二節 細胞移動性 9
第三節 中草藥癌症治療 10
第四節 Human Liver DnaJ-like protein (HLJ1) 10
一、 肺癌分期與治療 10
二、 HLJ1之轉錄調控 11
三、 血管新生試驗模式動物 12
第二章 流程圖 13
第三章 材料與方法 14
第一節 細胞株之選用 14
一、 人類非小細胞肺腺癌細胞(Human lung adenocarcinoma cell lines, CL1-0, CL1-5 ) 14
第二節 細胞培養液之配置及繼代 14
一、 培養液之配置 14
二、 細胞解凍 15
三、 細胞培養 15
四、 細胞繼代 15
五、 細胞冷凍保存 15
第三節 CL1-0/luc+ 細胞株之挑選與培養 16
一、 構築pPNT-luc質體 16
二、 細胞轉染 16
第四節 冷光報導基因分析 17
一、 細胞液萃取方法及冷光分析 17
二、 篩選穩定表現冷光細胞株( Select CL1-0/luc+ stable cell line) 17
第五節 選擇中草藥 18
一、 篩選中草藥萃取物 18
二、 藥劑之準備 18
三、 藥物濃度測試 (IC20) 18
第六節 細胞遷移分析 (Wound-healing assay) 19
第七節 血管新生試驗 (Angiogenesis assay) 19
一、 模式動物(Animal model)前處理 19
二、 中草藥萃取物前處理 19
三、 雞蛋絨尿膜血管新生試驗 (Chorioallantoic membrane assay) 20
第八節 細胞轉移能力分析 (Cancer cell migration assay) 20
第九節 3D 腫瘤球體培養 (3D spheroid invasion assay) 21
第十節 電泳速度變動分析(Electrophoretic mobility shift assay, EMSA) 21
一、 核蛋白萃取 21
二、 純化引子 22
三、 引子接合 (probe annealing) 22
四、 End-labeling of oligonucleotide 23
五、 Protein/Probe Binding 24
六、 Electrophoresis 24
第四章 結果 25
一、 成功篩選出CL1-0/luc+穩定表現細胞株 25
二、 測定細胞CL1-0之毒性 25
三、 中草藥影響肺腺癌細胞CL1-5生長能力 26
四、 中草藥影響血管新生能力 26
五、 中草藥影響肺腺癌細胞CL1-5轉移(Migration)試驗 26
六、 3D 腫瘤侵入試驗測試中草藥影響CL1-5侵入 能力 27
七、 Real-time PCR分析CL1-5細胞株HLJ1表現量 27
八、 HLJ1啟動子區域轉錄因子分析 27
九、 EMSA 28
第五章 討論 30
第六章 結論 33
實驗結果表圖 34
附圖 52
附表 54
54
參考文獻 56

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