臺灣博碩士論文加值系統

本實驗室先前研究發現，肺癌病人肺組織中的腫瘤細胞比正常細胞表現較高量的AhR，且肺腺癌細胞株也比正常肺細胞株表現較高量的AhR，故推測AhR可能與肺腺癌的發生（carcinogenesis）有關。另一方面，過去研究指出AhR與皮膚角質細胞等的細胞分化（differentiation）有關，並且由本實驗室之前研究知道，人類細支氣管上皮有表現AhR。因此本實驗的目的為：1) 利用RNA干擾技術，抑制肺腺癌細胞株H1355中AhR的表現，探討AhR在肺癌細胞中扮演的生理角色，2) 分析AhR在肺細支氣管上皮細胞分化中表現量之變化。我們首先建構一個AhR RNAi的DNA載體，轉染至H1355細胞株並經G418篩選後，挑選能持續穩定地表現AhR RNAi的stable clones，以定量即時同步聚合鏈鎖反應及西方墨點法確定stable clones中的AhR RNA和protein表現量受到抑制的程度達80﹪，並且AhR的轉錄活性也受到抑制，進一步以流式細胞儀分析細胞週期分布情形，發現AhR RNAi stable clones有較高的比例的細胞凋亡及G2/M期停滯情形。皮下注射4x106 AhR RNA i stable clones細胞至裸鼠背部，測量紀錄腫瘤的大小發現，測量開始後的前25天內，AhR RNA i腫瘤小於AhR wild-type與vector-only控制組，但第41天測量結束時AhR RNA i腫瘤雖然仍小於vector-only控制組，但與AhR wild-type控制組差異卻變的不明顯。以上結果推論，AhR可能與增加癌細胞的生長速度有關。另一方面，我們建構正常的人類肺細支氣管上皮細胞（small airway epithelial cell，SAEC）之細胞分化模式，來探討細胞分化過程中AhR表現量的變化。SAEC依細胞型態可將其分為細胞核質比（N/C ratio）較高的基底細胞（basal cells），和核質比較低的上皮細胞（epithelial cells），我們對SAEC處理高劑量（1mM）的Ca++來促使細胞分化，發現Ca++會促使基底細胞細胞比例增加而上皮細胞比例下降，進一步利用細胞免疫染色分析肺細支氣管上皮細胞各類型細胞的細胞標誌，結果顯示，未經處理組的上皮細胞主要表現cytokeratin 14（CK14），也會表現cytokeratin 7（CK7），但幾乎不表現Clara cell secretory protein（CCSP），而基底細胞主要表現CK14。當細胞經1mM calcium處理3天後，上皮細胞的CCSP表現明顯增加（p<0.05），並且基底細胞CK14的表現比例明顯增加（p<0.05）。此外我們發現此分化過程中AhR的RNA和 protein表現量皆有增加。當我們進一步對細胞處理AhR的配基（戴奧辛及benzo[a]pyrene）後發現，CYP1A1、 CYP1B1受誘導增加程度在分化的SAEC細胞比在未分化的SAEC細胞高出許多。綜合以上結論，AhR表現量的增加可能會促進肺腺癌細胞的生長，並且其表現及活性隨著肺細支氣管上皮細胞分化過程而增加。

Our previous studies indicated that aryl hydrocarbon receptor (AhR) expression was up-regulated in human lung adenocarcinoma (AD) tissues and cell lines. It suggested that AhR might play an important role in the development of lung adenocarcinoma. On the other hand, AhR expression was associated with differentiation in some normal cell types, such as keratinocytes. Previously we reported that AhR was present in the human bronchiolar epithelium. There are two objectives in this study: 1) to evaluate the function of AhR in lung adenocarcinoma cells with the RNAi technique, 2) to analysis the AhR expression level and activity during differentiation of human peripheral lung epithelial cells. We created a DNA vector containing human HU6 promoter followed by the AhR small interference sequence. This construct was transfected into lung AD cell lines H1355 and G418 resistant stable clones were selected. The quantitative real-time PCR and Western immunoblotting analysis showed that AhR mRNA levels in these stable clones were inhibited up to 80%. AhR RNAi stable clones showed decreased levels of cytochrome P4501A1 (CYP1A1) induction by TCDD, which was regulated by AhR. The increase in apoptosis and G2/M arrest was found in AhR RNAi stable clones. Tumorgenicity of these clones was evaluated by subcutaneously injection into flanking nude mice. At earlier days (25days) mice bearing AhR RNAi tumors had smaller and less numbers of tumors than mice bearing vector control tumors or wild types cells. But the difference disappeared at later days (41day). These data suggested that AhR might increase growth of <a href="http://www.ntsearch.com/search.php?q=cancer&v=56">cancer</a> cells. In the present study, we utilized human small airway epithelial cell (SAEC) as a model to monitor the AhR expression levels during differentiation of peripheral lung epithelial cells. Classified by cell morphology, the cultures of SAEC contained two celltypes: basal cells with high nuclei/cytoplasm ratio, and epithelial cells with low nuclei/cytoplasm ratio. Treatment with 1 mM calcium induced differentiation of SAEC. During differentiation, the proportion of basal cells increased, but that of epithelial cells decreased. Using the immunocytochemistry method, we found that epithelial cells mainly expressed cytokeratin 14 (CK14), cytokeratin 7 (CK7), but not Clara cell secretory protein (CCSP). Basal cells expressed cytokeratin 14 (CK14). After 3 days treatment with 1 mM calcium, expression of CCSP and CK14 was respectively increased in epithelial and basal cells (P<0.05). AhR mRNA and protein levels, measured with the real-time RT-PCR and Western immunoblot, were increased after calcium treatment for 3 days. It is well known that AhR regulates cytochromeP4501A1 (CYP1A1) and cytochromeP4501B1 (CYP1B1) gene expression. We found that AhR agonists, TCDD and BaP, -induced CYP1A1 and CYP1B1 levels were significantly increased during differentiation. In summary, increased AhR expression might increase the growth of lung adenocarcinoma cells. Increased AhR expression and activity was also found during differentiation of human peripheral lung epithelial cells.

目 錄 壹、 中文摘要 1 貳、 英文摘要 3 參、 縮寫表 5 肆、 前言 6 一、 肺癌 6 二、 戴奧辛受器（AhR）與TCDD、BaP的毒性 7 三、 RNA 干擾 9 四、 AhR與細胞週期（cell cycle）之關係 11 五、 AhR與細胞分化（cell differentiation）的關係 12 六、 呼吸系統 13 七、 肺上皮幹細胞（lung epithelial stem cell） 14 八、 研究動機 15 伍、材料與方法 17 一、 材料 17 1. 酵素 17 2. 試藥 17 3. 細胞株 18 4. 質體 18 5. 儀器 19 二、 實驗方法 19 1. 微量質體DNA的萃取 (Miniprep) 19 2. 中量質體DNA的萃取 (Midiprep) 20 3. 利用化學法製備勝任細胞 ( competent cell ) 21 4. RNA干擾的載體構築 22
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5. 細胞培養 26 6. 細胞轉殖作用（Transfection） 27 7. 經由G418篩選Stable clones 27 8. 細胞RNA的萃取 28 9. cDNA的合成 28 10. 定量PCR (quantitative real-time PCR) 29 11. 製備細胞均質液 31 12. BCA蛋白定量法 32 13. 西方墨點法 33 14. 細胞週期分析 34 15. 裸鼠皮下腫瘤生成能力 34 16. 免疫組織染色法 (Immunohistochemistry) 35 17. 免疫細胞染色法 (Immunocytochemistry) 35 18. 統計分析 36 陸、實驗結果 37 一、AhR RNA干擾載體的構築 37 二、篩選AhR RNA干擾之stable clones 39 三、分析AhR RNA干擾抑制AhR表現量的情形 41 四、AhR RNAi 對CYP1A1、CYP1B1表現量的影響 42 五、AhR RNAi 對細胞週期分布的影響 43 六、AhR RNAi對裸鼠腫瘤生成能力的影響 44 七、裸鼠腫瘤中AhR的表現情形 45 八、評估AhR RNAi stable clones的穩定性 46 九、Calcium對SAEC細胞分化的影響 47 十、Calcium與SAEC分化之劑量反應與時間反應關係 49
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十一、Calcium對SAEC細胞中AhR表現的影響 50 十二、Calcium對AhR、Arnt基因表現的影響 51 十三、Calcium對TCDD誘導CYP1A1、CYP1B1基因表現的影響 52 柒、結論 53 捌、討論 54 玖、圖表 59 圖一、檢測AhR RNA干擾序列是否接合至pcDNA3.1(-)/CMV-/HU6載體上 59 圖二、AhR RNA干擾載體示意圖 60 圖三、篩選AhR RNA干擾之stable clones 61 圖四、分析AhR RNA干擾抑制AhR表現量的情形 62 圖五、AhR RNAi 對CYP1A1、CYP1B1表現量的影響 63 圖六、AhR RNAi 對細胞週期分布的影響 64 圖七、AhR RNAi對裸鼠腫瘤生成能力的影響 65 圖八、裸鼠腫瘤中AhR的表現情形 66 圖九、評估AhR RNAi stable clones的穩定性 67 圖十、SAEC的細胞型態 68 圖十一、分析肺上皮細胞cell markers 69 圖十二、Calcium與SAEC細胞分化之劑量反應關係（dose-response） 70 圖十三、Calcium與SAEC細胞分化之時間反應關係（time-course） 71 圖十四、Calcium對SAEC細胞中AhR表現的影響 72 圖十五、Calcium對SAEC細胞中AhR、Arnt表現之影響 73 圖十六、Calcium是否影響SAEC細胞中的TCDD誘導CYP1A1、
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CYP1B1基因表現 74 表一、Calcium對SAEC細胞中cell markers表現的影響 75 壹拾、附圖 76 附圖一、AhR訊息傳遞路徑 76 附圖二、RNA干擾機制圖（果蠅model） 77 附圖三、呼吸系統構造 78 附表一、引子序列 79 附表二、肺上皮細胞的細胞標誌 80 附表三、肺上皮幹細胞及細胞譜系（cell lineages） 81 壹拾壹、參考文獻 82

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