由福馬林固定與石蠟包埋樣品中萃取蛋白質以發掘可能之生物標幟蛋白

Proteins Extracted from Formalin-Fixed and Paraffin-Embedded Samples for Biomarkers Mining

黃碧標(Bee-Piao Huang)

指導教授 ： 高紹軒

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00082

摘要

福馬林固定石蠟包埋標本廣泛使用在病理對癌症和疾病作檢查，但是很少也很困難去作進一步之研究。此研究主要是希望能建立從福馬林固定石蠟包埋formalin-fixed, paraffin-embedded (FFPE)標本切片中抽取蛋白質的方法，用於發現新的標幟蛋白。
目前主要使用加熱抗原復活技術heat-induced antigen retrieval (AR) technique的蛋白質萃取法已廣泛使用在福馬林固定石蠟包埋標本的免疫化學染色之相關分析。因此，本研究以加熱抗原復活 (AR)方法為基礎，先利用豬肝石蠟切片進行一系列實驗，建立蛋白質萃取之最佳參數。再使用已建立之最佳參數使用在人類的蠟塊標本之蛋白質萃取。
我們使用經兩位病理醫師確診為癌化細胞的福馬林固定石蠟包埋檢體，切片並使用不同的方法萃取蛋白質。評估萃取的效果採用兩種不同方式進行評估。第一，經本方法萃取之蛋白質以聚丙烯醯胺膠體電泳 (SDS-PAGE)進行分離接著以考馬氏亮藍染色 (Coomassie Brilliant Blue staining)，進行萃取蛋白式樣 (protein pattern) 評估。第二，將經過萃取後的樣本沉澱物以酒精脫水、二甲苯澄清後，重新以石蠟包埋並做成切片。重製之石蠟切片標本以蘇木紫伊紅染色進行萃取完成度 (completeness of extraction) 評估。依據一系列的萃取實驗結果發現，樣本經DNase處理後，接著以2% SDS/Tris萃取緩衝液進行加熱萃取之效果最好。此一蛋白質萃取式樣評估結果與石蠟切片標本以蘇木紫伊紅染色染色評估結果相印証。
因此，我們進一步利用此一萃取方法，進行膽管癌之標幟蛋白的相關分析。實驗結果發現比較膽管癌與正常膽管之蛋白質萃取式樣中，有七條顯著不同的蛋白質色帶，推測這些不同表現量的蛋白質可能與膽管癌有關。因此進一步使用膠內酵素消化與質譜鑑定分析，企圖鑑定出這些蛋白質之身分 (protein identity)。鑑定結果顯示熱壓力蛋白HSP60與CCKAR似乎為膽管癌可能之新穎標幟蛋白。進一步的免疫組織染色更證明HSP60與CCKAR與癌細胞之移轉也有密切的關係。
本研究成功地從福馬林固定石蠟包埋檢體中，建立一個萃取蛋白質的方法。利用此一萃取方法確實可以從福馬林固定石蠟包埋檢體中，萃取出特殊的蛋白質式樣，而且可以從此一蛋白質式樣的比較，得到腫瘤組織與正常組織表現不同的蛋白。這些具有差異性表現的蛋白質，經進一步的實驗驗證確實可能成為新穎之癌細胞標幟蛋白。

關鍵字

福馬林固定石蠟包埋 ； 癌 ； 熱誘導抗原取得技術 ； 標幟蛋白

並列摘要

Formalin-fixed and paraffin-embedded (FFPE) samples are widely used in pathological examination for carcinoma and various diseases, but the FFPE sections are rare and difficult to be further investigated. The present study aimed to establish an extraction approach to obtain proteins from FFPE samples, which could be useful in mining potential protein markers. A satisfactory protocol of the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival FFPE tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE pig liver tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human。Carcinoma FFPE samples, confirmed by two pathological doctors and then treated with different methods to extract proteins. To evaluate the effects of each extraction approach on FFPE sample, the treated FFPE slices were further dehydrated with ethanol, reacted with xylene, embedded with paraffin and sectioned. The efficiency of each extraction approach was both evaluated by CBB-stained protein profile on the SDS-acrylamide gel and the H-E stained profile of re-fixed and embedded samples. The CBB-stained protein profiles revealed that DNase treatment following 2% SDS/Tris heating extraction was the most effective approach to extract proteins from FFPE samples in this study. These findings were consistent with the HE-stained profiles of treated FFPE samples. Moreover, the CBB-stained protein profiles presented seven protein bands with different levels among CHC and normal biliary duct. The proteins with different level might be potential markers to distinguish CHC from the normal biliary duct. The present study established a protein extraction approach combining DNase treatment and SDS/Tris heating extraction for FFPE samples. By using the approach, proteins extraction for FFPE samples should be more effectively, the findings which may be useful and helpful for mining potential carcinoma markers from FFPE sections.

並列關鍵字

Formalin-fixed and paraffin-embedded ； carcinoma ； protein marker

參考文獻

1.Chen J, Balgley BM, DeVoe DL, Lee CS. Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis. Anal Chem . 2003；75:3145~52.

2.Saravanan RS, Rose JKC . A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics. 2004；4:2522~32.

3.Shi SR, Key ME, Kalra KL . Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem . 1991；39:741~8.

5.Gown AM . Unmasking the mysteries of antigen or epitope retrieval and formalin fixation. Am J Clin Pathol . 2004；121:172~4.

6.Taylor CR, Cote RJ. Immunomicroscopy. A Diagnostic Tool for the Surgical Pathologist. 3rd ed. Philadelphia, Elsevier Saunders.