近十年來在台灣各種癌症中，肺癌的發生率及死亡率都是排名第一位；其中肺腺癌在最近二十年已逐漸增多，已成為最常發現的肺癌種類。肺癌的治療以化學治療為主， 大部分的作用在於將DNA破壞，但是反應率仍不理想。一般認為是因為DNA的修補功能，使得癌症細胞對化學治療有抗藥性。因此本實驗的目的在探討將DNA修復蛋白之一的Mre11默化後，對肺癌細胞的生長是否會造成影響。
本實驗第一部分為將57位罹患肺腺癌患者的標本，做成組織微陣列後，針對Mre11染色。並統計各項臨床變數，探討其表現的高低與生存率是否有影響。第二部分是利用shRNA將A549肺癌細胞的Mre11基因默化，再以載有空白載體的A549細胞做為對照組。之後進行XTT法及軟瓊脂細胞群落生長實驗來比較細胞生長有無差異；另以流式細胞儀探討其細胞週期有無變化。最後將A549細胞植入裸鼠皮下，比較腫瘤形成的大小。
在臨床的統計方面，患者沒有淋巴結與遠處轉移及有較高表現的Mre11，都代表有較好的存活率。進一步統計發現，只有淋巴結與遠處轉移才是預後的獨立因子，而Mre11的表現並不是。 在A549細胞的實驗部分，發現將Mre11的基因默化後會造成細胞的生長明顯變慢；且在細胞的週期上發現G0/G1及S期的比例上升，而G2/M期的比例下降。在裸鼠的實驗，也有發現腫瘤的生長明顯變慢的現象。基於上述實驗，抑制Mre11的表現可以造成腫瘤生長變慢及提供另一個治療肺癌的途徑。

In recent decade, lung cancers had the highest incidence and mortality rate among all cancers in Taiwan. Among lung cancers, adenocarcinoma was the most frequent type. The chemotherapy was still the main choice in treating lung cancer by the mechanism of destroying DNA, but the response rate kept low. The function of DNA repair makes cancer cells resistant to chemotherapy. Therefore, this study focused on the effect of cancer cell growth by silencing Mre11.
The first part of this study was to make a tissue microarray consisting of adenocarcinoma from 57 patients. Immunohistochemistry staining for Mre11 was done. The correlation of Mre11 expression and clinical variables with survival was analyzed. The second part was tried to knockdown Mre11 in A549 cell by shRNA. Another A549 cell line containing empty vector was selected as control group. These cell lines were then ready for XTT method, soft agar colony formation assay, flow cytometry and nude mice assay.
In the clinical data, the absence of lymph node and distant site metastasis were good prognosis factor for longer survival. Although the high expression on Mre11 had longer survival, this variable was not a true independent factor. On XTT method and soft agar colony formation assay, the A549 cells with Mre11 knockdown had a slower proliferation and fewer colony numbers, respectively. The cell cycle demonstrated an elevated G0/G1 and S phase and depressed G2/M phase in A549 cells with Mre11 knockdown. The tumor arising from A549 cells with Mre11 knockdown in the nude mice also had a smaller size. Based on the above study, inhibition of Mre11 may result in a reduction of tumor growth and provide another choice to treat lung cancer.

論文審定書…………………………………………………………… i
誌謝…………………………………………………………………… ii
中文摘要……………………………………………………………… iii
英文摘要……………………………………………………………… iv
碩士論文正文 ……………………………………………………… 1
緒論…………………………………………………………………… 2
一、肺癌的病因與治療…………………………………………… 2
二、DNA 雙股斷裂時的修補與Mre11 蛋白的功能與構造…… 2
三、因為修補蛋白突變所產生的病變 ………………………… 4
四、目前肺腺癌的治療與困境及展望 ………………………… 5
五、研究目標……………………………………………………… 5
研究材料與方法……………………………………………………… 7
一、臨床資料……………………………………………………… 7
二、組織微陣列與免疫組織化學染色…………………………… 7
三、組織微陣列的免疫組織化學染色的判讀………………………7
四、比較Mre11蛋白在正常肺組織細胞與各種肺癌細胞的表現…8
五、以短夾核醣核酸(shRNA)來降低肺癌細胞中的Mre11蛋白… 8
六、細胞存活率分析……………………………………………… 9
七、XTT 法………………………………………………………… 9
八、軟瓊脂細胞群落生長實驗………………………………………9
九、以流式細胞儀檢視細胞週期的變化………………………… 10
十、腫瘤細胞植入裸鼠皮下的生長實驗………………………… 10
十一、統計方法…………………………………………………… 11
研 究 結 果……………………………………………………… 12
一、肺腺癌病患Mre11 的表現與臨床資料分析…………………12
二、默化的Mre11 蛋白對肺腺癌細胞株及活體腫瘤的影響……13
結 果 討 論……………………………………………………… 15
未 來 研 究 方 向………………………………………………… 17
參 考 文 獻………………………………………………………… 34
附 錄……………………………………………………………………38

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