臺灣博碩士論文加值系統

在1984年，Nakamura等從肝臟部分切除的大鼠血清中發現了一種新的生長因子，此生長因子具有促進肝臟細胞生長和DNA合成的特性，因此將之命名為肝細胞生長因子。肝細胞生長因子本身也是強而有力的細胞素，可以誘發細胞的移動和細胞型態的改變。此因子主要是由間質細胞所製造，以旁泌素的形式作用在鄰近的上皮細胞，藉由活化細胞膜上的受體Met，來啟動下游酪胺酸基酶的訊息傳遞路徑。先前的研究已經證實過度表現肝細胞生長因子和Met與人類腫瘤的形成和轉移有關，在癌症病患常象徵會有預後不良的結果。
本實驗室先前已經發現，吸煙引發肺腺癌的病患肝細胞生長因子mRNA都有過度表現的情形，而肝細胞生長因子主要表現在腫瘤周圍的正常組織。肺腺癌是人類肺癌中最常發生的形式。隨著吸煙人口的增加，吸煙已經變成肺癌形成最主要的風險因子，特別是肺腺癌。在人類的肺臟組織，藉著原位雜合染色，本研究結果發現肝細胞生長因子mRNA表現在第二型肺泡細胞，如同肝細胞生長因子會表現在A549細胞株一般。體外培養第二型肺泡細胞，我們證明香煙中的尼古丁會誘發細胞表現肝細胞生長因子。活體實驗中，我們建立小鼠吸煙的動物模式，分別吸煙10個星期和20個星期來評估肝細胞生長因子的表現。在吸煙小鼠中可見到第二型細胞有顯著地增生，肝細胞生長因子的表現也向上調控。我們也發現尼古丁會誘發第二型細胞FAK的轉錄，在吸煙小鼠肺臟組織中FAK蛋白的表現有增加之情形。根據這些結果，我們推論尼古丁會誘發第二型細胞表現肝細胞生長因子。

Hepatocyte growth factor (HGF) was first purified from serum of hepatectomized rats by Nakamura et al. in 1984, based on it’s ability to promote liver cell growth and DNA synthesis. HGF is also a potent cytokine to induce cell motility, and morphogenesis. It is mainly secreted by mesenchymal cells and acts on a wide variety of epithelial cells through binding of membrane receptor c-Met and activation on tyrosine kinase signaling cascade. The previous studies demonstrated that overexpression of HGF and c-Met often correlated with human tumorigenesis and metastasis, in particular, with the poor prognosis of cancer patients.
Previously, our lab discovered that HGF mRNA was overexpressed in nontumor lung tissue of adenocarcinoma patients induced by cigarette smoke. Adenocarcinoma is a common form of lung cancer. As the increase of smoking population, cigarette smoking becomes a leading risk factor for lung cancer development, especially for lung adenocarcinoma. By in situ hybridization, we found in this study that HGF mRNA was expressed in type II alveolar epithelial cells as well as A549 cell line. In vitro, we showed that cigarette smoke derivative, nicotine, induced HGF expression in primary culture of type II cells. In vivo, we set up an animal model of mouse to evaluate cigarette smoking on HGF expression. After ten- and twenty-week cigarette smoke exposure, proliferation of type II cells increase significantly in cigarette smoking mice and HGF expression was also up-regulated. We also found that nicotine induced FAK transcription in type II cells and increased in cigarette smoking mice. According to these results, we suggest that nicotine could induce HGF expression in type II cells.

中文摘要...................................................................................................................... Ⅰ
英文摘要....................................................................................................................... Ⅱ
縮寫對照表................................................................................................................... Ⅲ
目錄.............................................................................................................................. Ⅴ
圖表目錄...................................................................................................................... Ⅶ
第一章 緒論
第一節 文獻回顧................................................................................................. 1
一、肝細胞生長因子..................................................................................... 1
二、FAK簡介.............................................................................................. 9
三、第二型肺泡細胞.................................................................................... 10
四、肺癌簡介................................................................................................ 12
第二節 研究動機.................................................................................................. 14
第二章 材料與方法
第一節 實驗動物與細胞株................................................................................ 16
第二節 肺癌細胞株的培養............................................................................... 16
一、細胞株的種類及細胞培養.................................................................... 16
二、細胞株冷凍保存、計數及解凍............................................................ 16
第三節 初代培養第二型肺泡細胞..................................................................... 17
第四節 萃取RNA….......................................................................................... 18
第五節 反轉錄聚合酶連鎖反應......................................................................... 19
一、反轉錄反應.......................................................................................... 19
二、聚合酶連鎖反應................................................................................. 19
第六節 即時定量聚合酶連鎖反應................................................................... 20
一、即時定量聚合酶連鎖反應的特性....................................................... 20
二、即時定量聚合酶連鎖反應的操作....................................................... 21
第七節 聚丙烯醯胺膠體電泳............................................................................. 21
第八節 西方墨點法.......................................................................................... 22
第九節 組織切片染色....................................................................................... 23
一、特殊柏氏染色....................................................................................... 23
二、免疫組織化學染色............................................................................... 23
三、免疫細胞化學染色............................................................................... 24
四、原位雜交............................................................................................... 24
第十節 原核表現載體之構築..................................................................... 25
一、目標片段的增幅與TA cloning............................................................ 25
二、轉形作用......................................................................................... 26
三、抽取小量質體DNA......................................................................... 26
四、限制酶反應...................................................................................... 26
五、菌種甘油保存...................................................................................... 27
第十一節 重組蛋白之製備與純化................................................................. 27
一、pET32a重組蛋白表現載體之構築..................................................... 27
二、純化PCR產物..................................................................................... 27
三、純化膠體中之DNA片段.................................................................... 27
四、DNA黏合反應.................................................................................... 28
五、轉形至蛋白表現之勝任細胞............................................................... 28
六、pET32a重組蛋白之條件測試........................................................... 28
七、pET32a重組蛋白之大量表現........................................................... 29
八、pET32a重組蛋白之純化.................................................................. 29
第十二節 抗體的製備........................................................................................ 30
一、免疫抗原之製備................................................................................ 30
二、蛋白質定量........................................................................................... 30
三、小鼠之免疫反應................................................................................. 30
四、酵素結合免疫吸附反應..................................................................... 31
第三章 結果
第一節 確認第二型肺泡細胞表現肝細胞生長因子......................................... 32
第二節 初代培養第二型肺泡細胞..................................................................... 32
第三節 分析尼古丁刺激第二型肺泡細胞表現HGF......................................... 33
第四節 偵測尼古丁受體的表現......................................................................... 34
第五節 在活體中分析尼古丁對於第二型肺泡細胞表現HGF的影響........... 35
第六節 分析SP-B、FAK在吸煙小鼠與未吸煙小鼠肺泡組織的表現........... 36
第四章 討論................................................................................................................. 38
第五章 結論.................................................................................................................. 44
參考文獻........................................................................................................................ 45
圖表................................................................................................................................ 63
附表與附圖.................................................................................................................... 80
附錄................................................................................................................................ 87

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