利用蛋白質體技術鑑定於胃癌上皮細胞中缬絡胺酸蛋白質之交互作用體

Abstract

過去本實驗室利用蛋白質體學技術(proteomics approach)以及免疫組織染色，對照未感染幽門螺旋桿菌以及感染幽門螺旋桿菌的AGS 胃腺癌細胞，發現許多經幽門螺旋桿菌感染後表現量會上升的蛋白質，有些蛋白質為已知可能之治癌因子，其中又以缬絡胺酸蛋白質valosin containing protein (VCP)這個重要蛋白質表現差異最大，稍後利用mRNA微陣列結合蛋白質體學，得到許多可能受VCP調控之宿主細胞因子。至今缬絡胺酸的研究領域當中，發現許多種類癌症都有大量表現的情形，並在病人組織中的缬絡胺酸含量可以做為癌症預後狀況的指標。然而缬絡胺酸與癌症發展之間的關係尚未完全研究清楚，本論文的實驗目的希望利用蛋白質體學技術，研究AGS 胃腺癌細胞大量表現，利用免疫沉澱(immunoprecipitation)方法，觀察細胞質中與缬絡胺酸交互作用的蛋白質並進一步探討之。 嘗試過不同的轉染方式後，我們採用帶有表現綠色螢光蛋白且尾端接上flag tag缬絡胺酸質體感染胃上皮細胞，使之大量表現缬絡胺酸，也由於在細胞質中表現，因此利用接有辨認flag tag抗體的凝膠珠去把跟缬絡胺酸於胃上皮細胞正常生理下有交互作用蛋白質一併抓下，此過程稱之為共免疫沉(co-immunoprecipitation)，抓下來的蛋白質複合體，則採取蛋白質體學的方式，經過一維電泳展開複合體組成蛋白質合併液相層析偶合串聯式質譜儀來分析與單有表現綠色螢光蛋白的胃上皮細胞也進行免疫沉澱的相比較，扣除掉非專一性結合的蛋白質後，我們找到有158個有差異的蛋白質，依蛋白質功能粗略的分為:1.蛋白質合成與蛋白質摺疊之相關因子; 2. 細胞骨架蛋白質; 3.細胞代謝酵素; 4.轉錄與轉譯相關因子;5其他。參考文獻之後，我們選出其中的30個與癌症相關蛋白質，並利用訊息傳遞資料庫的分析將鑑定的結果進一步推測出可能之訊息傳遞網路，其中較有可能藉由缬絡胺酸的分子導護幫助進行傳導的訊息途徑有二，一為腫瘤抑制因子p53與DNA修復的訊息傳導，二為因缺氧環境而產生的代謝途徑的適應。然而這些訊息傳導途徑皆導向更上游經PKB/Akt訊息傳導所共同調控，未來可望將這些結果做更進一步的討論，以利於在VCP於胃上皮細胞中參與調控的致病機制進一步的發現。

By using proteomics approaches and immunohistocehmistry in the past, our laboratory found that the protein expression level of many cancer-related factors were elevated in AGS cells after H. pylori infection, and one important factor was valosin-containing protein (VCP). Recently studies are showed that VCP overexpresses in almost every kind of cancer, and the level of VCP in cancerous tissues is a prognosis marker for cancer diseases. However it is not a clear relationship between VCP expression level and cancer development. The specific aim of this thesis is to investigate the interacting proteins with VCP. By overexpressing VCP in AGS cells and co-immunoprecipitating the whole complexes, further the complexes are identified by using proteomics approaches. The number of identification proteins deducting from nonspecific binding proteins is 158. These were classified as transcription and translation-related proteins and folding-related proteins, cytoskeleton proteins, metabolic enzymes, cell communication/ signal transduction-related proteins on the basis of their molecular functions. After literature search, we select 30 proteins which are more significant in cancer development and H. pylori infection. The Metacore help in linking these proteins and producing possible signal network. There are two depended on the analysis: (1) ATM-Chk2-p53 cascade and (2) VHL-HIF1 pathway. VCP as a molecular chaperone may promote degradation of p53 and VHL-HIF and therefore cells are anti-apoptosis. And the two pathways are both PKB/Akt downstream signal pathway. VCP was also documented to play an essential role in Akt downstream signaling, so we proposed the aforementioned findings were caused by the facilitation of Akt signaling in VCP-overexpressed cells. Therefore, we hope the complete interactome can be identified so that we can study the detail mechanism of VCP in cancer development.