Protein Extraction for Potential Cholangiocarcinoma Biomarkers Mining from Formalin-Fixed and Paraffin-Embedded Samples

由福馬林固定石蠟包埋樣品中萃取蛋白質的方法以發掘可能的膽管癌標幟蛋白

黃碧標(Bee-Piao Huang) ； 王威鈞(Wei-Jiun Wang) ； 蘇熙元(Si-Yuan Su) ； 徐瑋佑(Wei-Yu Hsu) ； 高紹軒(Shao-Hsuan Kao)

《中山醫學雜誌》 21卷1期 (2010/03) Pp. 35-45

https://doi.org/10.30096/CSMJ.201003.0003

摘要

福馬林固定之石蠟包埋樣品廣泛運用於各式疾病之病理檢定，然而經過福馬林固定之石蠟包埋樣品卻難以再進行進一步的分析。主要的原因除福馬林固定所造成的蛋白質交聯之外，石蠟也會對相關的質譜分析產生干擾。因此，本研究企圖建立一種可以有效自福馬林固定之石蠟包埋樣品中萃取出蛋白質的方法，以利後續相關的傳統生化分析與蛋白質體研究。我們的研究結果顯示二甲苯脫蠟是很重要的步驟，且二甲苯脫蠟並復水後的樣品，經DNase酵素處理再以SDS/Tris緩衝液進行蛋白質萃取，於聚丙烯醯胺膠體電泳呈現出清楚的分離效果。接著利用這個蛋白質的萃取方法，可以在解析度有限的聚丙烯醯胺膠體分析中，顯現出不同腫瘤樣品：包括膽管癌、大腸癌與肝癌，具有不同的蛋白質表現。此外，透過西方墨點法，也可以清楚地辨識出已知的腫瘤標幟蛋白，如CK-8/18、CK-7與glypican-3。據此，我們推測此一蛋白質萃取法可結合現有之生化免疫分析與蛋白質體研究，運用於發現可能的新型腫瘤標幟蛋白以及更進一步的癌化機制。

關鍵字

福馬林固定與石蠟包埋 ； 膽管癌 ； 標幟蛋白

並列摘要

Formalin-fixed and paraffin-embedded (FFPE) samples are widely used in pathological examination for carcinomas and various diseases. Therefore, developing an efficient protein extraction approach would make it possible for many diagnostic FFPE samples to be mined for biomarkers. The present study aimed to establish a method of extracting proteins from FFPE samples that would make it also possible to perform subsequent biochemical and proteomic analyses and be useful in the search for potential biomarkers of cholangiocarcinoma (CHC). We found that proteins could be effectively extracted from FFPE samples by DNase treatment followed by 2% SDS/Tris heating extraction, as evidenced by SDS-PAGE, morphological examination and Western blot analysis. The proteins extracted this way displayed different protein patterns differentiating between CHC, colon cancer (CC) and hepatocellular carcinoma (HCC). These proteins expressed at different levels might be used as potential biomarkers of CHC following further biological validation, and they might be used to help up gain a better understanding of the carcinogenesis of CHC.

並列關鍵字

Formalin-fixed and paraffin-embedded ； cholangiocarcinoma ； biomarker