藉由蛋白質體學分析子宮頸癌與轉移相關之生物標記分子

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研究生：	林逸婕 Lin, Yi Chieh
論文名稱：	藉由蛋白質體學分析子宮頸癌與轉移相關之生物標記分子 Proteomic Analysis of Metastatic Biomarkers in Cervical Cancer
指導教授：	詹鴻霖 Chan, Hong Lin
口試委員:	王浩文 Wang, Hao-Ven 周秀專 Chou, Hsiu-Chuan
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2015
畢業學年度：	103
語文別：	中文
論文頁數：	96
中文關鍵詞：	轉移、子宮頸癌、PGRMC1 、蛋白質體學
外文關鍵詞：	metastasis, cervical cancer, PGRMC1, proteomics
相關次數：	點閱：2 下載：0
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根據最新台灣衛生署102年統計，國人死大死因榜首為惡性腫瘤，而子宮頸癌位居於女性癌症中第七位。而根據WHO之2011年統計子宮頸癌為女性癌症中發生率第三高之癌症，每年約有530,000新診斷子宮頸癌之個案，且一半會因此而死亡。癌症死亡率如此高之原因主要由於癌症組織除了會在原部位發展外，到晚期會進行遠端轉移至其他維生器官如肝、腦、肺等等，並極有可能造成病患死亡。而子宮頸癌早期之五年存活率相當高，但當轉移一旦發生時，五年存活率變大幅降低。因此，在本論文研究中將具有不同轉移能力之子宮頸癌細胞株利用蛋白質體學之方式，探討轉移相關機制以及找到轉移相關之生物標記分子。
在本實驗中我們利用相同基因背景之HeLa與較具有侵襲能力之HeLa-I5做研究並希望找出具有潛力之轉移相關之蛋白。我們利用二維差異電泳(2D-DIGE)、基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF MS)找出差異之蛋白質。在結果中共鑑定出68個蛋白質具有差異，並利用西方點墨法(western blot)驗證。
在這些差異蛋白質中，我們選出一個較有潛力之蛋白-progesterone receptor membrane component1(PGRMC1)做後續實驗。並利用核醣核酸干擾技術(RNAi)使PGRMC1基因沉默。我們利用Transwell migration、傷口癒合實驗來觀察其蛋白對細胞移動能力之影響；Transwell invasion觀察細胞對細胞侵襲能力之影響，驗結果可以得知，PGRMC1對於子宮頸癌細胞之移動、侵襲能力以及對於細胞增生的速度有正面影響。本實驗利用蛋白質體學分析法找出在子宮頸癌轉移具有差異之蛋白質，並找出許多參與癌細胞侵入與轉移相關之潛力蛋白如PGRMC1，並希望能提供子宮頸癌與轉移相關的生物標記分子。

In Taiwan, Cervical cancer (CC) is the 7th leading cause of death in female cancer. Moreover, according to the WHO's report in 2011, cervical cancer is the third highest cancer incidence in female, around 530,000 new cases of cervical cancer were diagnosed each year with high death rate. The reason for a high mortality of cervical cancer is mainly due to cancer metastasis. When cervical cancer is diagnosed in early stage, the five-year survival rate is very high, but once metastasis occurs, the five-year survival rate reduced significantly. Thus, to manage cervical cancer, finding metastasis biomarkers and understanding the mechanisms are critical. In our current study, we used a pair of cervical adenocarcinoma cell lines, HeLa and its invasive partner, HeLa-I5, as a model system to examine invasive mechanism and find out the potential cellular targets associated with metastasis. We use two-dimensional differential gel electrophoresis difference (2D-DIGE), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify differentially expressed proteins between HeLa and HeLa-I5. Our proteomics study revealed that 68 proteins show significant difference between the two lines and some of the identified proteins have been validated through immunoblotting. Further study by using RNA interference (RNAi) technique, we found progesterone receptor membrane component1 (PGRMC1) protein is a key player in response to cervical cancer invasion, migration and proliferation. Our data demonstrated that the invasive ability and the migration ability decreased for 2-fold and 1.7-fold, respectively, in the process of PGRMC1 knockdown. To sum up, PGRMC1 has been evidenced to modulate cervical cancer migration, invasion and proliferation. Our data also provided useful diagnostic markers and therapeutic candidates for the treatment of cervical cancer invasion and migration.

Abbreviations    5
中文摘要    9
Abstract    10
壹、    文獻探討    11
一、    子宮頸癌(cervical cancer, CC)    11
(一)、    子宮頸癌之流行病學    11
(二)、    子宮頸癌分類    12
(三)、    子宮頸癌臨床分期    13
(四)、    子宮頸癌之危險因素    15
(五)、    臨床治療方式    15
二、    癌症轉移    15
三、    癌症轉移與子宮頸癌    17
四、    蛋白質體學    18
(一)、    簡介    18
(二)、    研究蛋白質體學之重要性    20
(三)、    二維凝膠電泳(two-dimensional gel electrophoresis)    20
(四)、    二維差異電泳(two-dimensional difference gel electrophoresis, 2D-DIGE)    23
(五)、    質譜儀    27
(六)、    蛋白身分鑑定    33
(七)、    蛋白質體學之研究範疇    35
五、    研究目的    37
貳、    材料與方法(Material and Methods)    38
一、    藥品與材料來源：    38
(一)、    細胞株來源：    38
(二)、    藥品與資料來源(表四)    38
二、    細胞株與培養方式    39
(一)、    細胞株與培養方式    39
(二)、Transwell invasion篩選細胞    39
三、    二維差異電泳分析與影像分析    40
(一)、    蛋白質樣品製備與定量    40
(二)、    蛋白質螢光標定    40
(三)、    等電點聚膠電泳 (isoelectric focusing, IEF)    41
(四)、    十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)    42
(五)、    分析蛋白質點與找出具有表現差異之蛋白    43
(六)、    膠染色    45
四、    蛋白質身分鑑定    45
(一)、    膠內酵素水解蛋白質(In-gel digestion)    45
(二)、    質譜分析    46
(三)、    搜尋資料庫已鑑定蛋白質之身分    46
五、    表現蛋白質之確認(Validation)    47
六、    小干擾核酸(small interfering RNA；siRNA)    49
七、    功能性分析(functional assay)    49
(一)、    基底膜基質侵犯能力與移動能力試驗(Transwell migration and invasion assay)    49
(二)、    傷口癒合實驗(wound healing assay)    50
(三)、    細胞存活率與細胞增長率(MTT proliferation assay)    51
參、    實驗結果    52
一、    HeLa與HeLa-I5細胞株的侵襲能力與特性之差異    52
二、    分析具有轉移能力差異之人類子宮頸癌蛋白質表現二維差異電泳分析    55
三、    二維差異電泳分析鑑定出之具有表現差異之蛋白質    57
四、    蛋白質修飾後其蛋白質位置與功能性分類    61
五、    以西方點墨法確認所鑑定之蛋白    62
六、    PGRMC1與子宮頸癌侵襲能力與移動能力之關聯    65
肆、    討論    73
伍、    結論    79
陸、    參考資料    80
柒、    附錄    87

                                

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