臺灣博碩士論文加值系統

PCTAIRE kinases 1 (PCTK1)在肺癌組織中具有高表現量，但PCTK1在癌症發展中所扮演的角色目前仍未知，它在神經細胞中會參與蛋白質分泌途徑以及神經細胞的生長與爬行。故本研究的目的是利用蛋白體學的方法找出PCTK1在肺癌細胞中影響的分泌蛋白，並藉此探討其在癌症發展所扮演的角色。我們利用dimethyl標定法結合液相層析串聯質譜儀 (LC-MS/MS)進行蛋白體學分析PCTK1所影響的分泌蛋白。我們收集H1299 PCTK1暫時敲減細胞與無敲減的對照組的分泌蛋白進行系統性分析，在兩次獨立實驗中鑑定出50個被PCTK1影響的蛋白，從中我們挑了annexin A2和extracellular matrix protein 1以西方墨點法進行驗證，兩者的分泌會受到PCTK1所抑制，但是mRNA表現量並不受影響。其次，我們也發現這50個蛋白質中，分泌上升者多數帶有外泌的signal peptide，而下降者多為細胞質或核的蛋白。另外，PCTK1敲減細胞的條件培養基對細胞的爬行能力沒有影響。總之，我們目前已證實在肺癌細胞中抑制PCTK1的表現確實會改變蛋白質的分泌，後續希望可以找到PCTK1如何藉由分泌蛋白去影響肺癌的進程與發展。

PCTAIRE kinase 1 (PCTK1) is highly expressed in lung cancer tissues. However, its role in cancer development has not been explored. In neuronal cells, PCTK1 is involved in vesicular transport, exocytosis, and neurite growth and migration. We hypothesize that PCTK1 may also influence secretory machinery in cancer cells. We applied a proteomics approach using dimethyl labeling and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to analyze the secretory proteins affected by PCTK1. First, the secretory proteins of H1299 PCTK1 transient knockdown cells were collected and analyzed with LC-MS/MS. In two independent experiments, we identified 50 proteins differentially secreted in PCTK1 knockdown cells. Then we used Western blotting to validate the secretory levels of two proteins, annexin A2 and extracellular matrix protein 1, which confirmed the results from LC-MS/MS. In addition, we found that many proteins with elevated secretion levels had signal peptide for secretion, whereas those with decreased levels were mostly cytosolic or nuclear proteins. In a functional study, the conditioned medium from PCTK1 knockdown cells did not increase cell migration. In conclusion, we have demonstrated that PCTK1 in lung cancer cells can regulate protein secretion, which may be important for cancer development or progression.

目錄
指導教授推薦書…………………………………………………………...
論文口試委員會審定書…………………………………………………...
誌謝.................................................................. iii
中文摘要 .................................................. iv
Abstract ........................................... v
目錄...................................................... vi
表目錄 ........................................... ix
圖目錄 ................................................... x
1. 文獻回顧 ............................................... 1
1.1 肺癌 ......................................................... 1
1.2 PCTAIRE kinases 1 (PCTK1) ................................................... 1
1.2.1 PCTAIRE kinases (PCTKs) ............................................ 1
1.2.2 PCTK1 ........................................................... 3
1.2.3 與PCTK1 有交互作用的蛋白 ...................................... 4
1.2.4 PCTK1 的生物功能 ........................................................ 6
1.3 蛋白分泌路徑 ........................................................ 9
1.4 Cancer secretome ................................................... 10
1.5 實驗室先前研究 ........................................................... 11
2. 實驗假說 ............................................................................. 13
3. 研究目的 ................................................................. 14
4. 材料與方法 ................................................ 16
4.1 細胞培養 ................................ 16
4.2 細胞轉染 ...................................... 16
4.3 條件培養基的收集 ............................. 16
4.4 分泌蛋白萃取 .................................... 17
4.5 細胞裂解液萃取 .......................... 17
4.6 SDS-PAGE .................................... 17
4.7 西方墨點法 ...................................... 18
4.8 LC-MS/MS in dimethyl labeling .................... 18
4.9 RNA 萃取 ...................................... 19
4.10 反轉錄定量聚合酶連鎖反應 (quantitative reverse
transcription PCR ; qRT-PCR) ...............................19
4.11 Wound healing assay ................................. 20
5. 結果 ...................................................... 21
5.1 抑制PCTK1 表現的H1299 細胞不會增加TIMP-1 的分泌量 ............................... 21
5.2 抑制PCTK1 表現會改變分泌蛋白的圖譜 ........................... 22
5.3 以西方墨點法確認蛋白質體學的結果 ................................. 23
5.4 Annexin A2 和 ECM1 的mRNA 表現量 ............................ 24
5.5 抑制PCTK1 表現後的分泌蛋白並不會影響的細胞爬行的能力......................... 24
6. 討論 ......................................................... 26
參考文獻 ............................ 30
表...................................... 36
圖............................................. 48

表目錄
表一、從SDS-PAGE 的差異條帶鑑定出的蛋白質 ............................. 36
表二、H1299 PCTK1 暫時敲減細胞分泌蛋白以dimethyl labeling 進
行LC-MS/MS 定量分析後，具有差異性的蛋白 (第一次) ................ 37
表三、H1299 PCTK1 暫時敲減細胞分泌蛋白以dimethyl labeling 進
行LC-MS/MS 定量分析後，具有差異性的蛋白 (第二次) ................ 39
表四、表二和表三交集的27 個分泌量上升蛋白 ................................ 41
表五、表二和表三交集的23 個分泌量下降蛋白 ................................ 42
表六、表四分泌上升蛋白的生物途徑分析 .......................................... 43
表七、表五分泌下降蛋白的生物途徑分析 .......................................... 44
表八、表二和表三聯集的140 個分泌量上升蛋白的生物途徑分析 .. 45
表九、表二和表三聯集的152 個分泌量下降蛋白的生物途徑分析 .. 46
圖目錄
圖一、抑制PCTK1 表現在H1299 肺癌細胞不會增加TIMP-1 的分泌........................ 48
圖二、綜觀兩次利用dimethyl labeling 和LC-MS/MS 分析H1299 細
胞暫時抑制PCTK1 表現所影響的分泌蛋白 ........................................ 49
圖三、驗證在H1299 細胞中抑制PCTK1 表現會促進annexin A2 和ECM1 的分泌 ............... 50
圖四、在H1975 細胞中抑制PCTK1 表現會不會影響annexin A2 和ECM1 的分泌 ................. 51
圖五、抑制H1299 細胞PCTK1 的表達不會影響annexin A2 和ECM1 的mRNA 表現量 ............. 52
圖六、抑制H1975 細胞PCTK1 的表達不會影響annexin A2 和ECM1 的mRNA 表現量 ............ 53
圖七、在H1299 細胞中抑制PCTK1 表達所分泌的蛋白並不會影響細胞爬行的能力 ................ 54
圖八、在H1975 細胞中抑制PCTK1 表達所分泌的蛋白並不會影響細胞爬行的能力 ............. 55

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