| 摘要: | 人類的唾液中含有豐富的蛋白質，而唾液樣品的取得為非侵略性，因此本論文以確立所發展的高效二維膠體電泳技術，進而探討應用於流行病學或臨床上診斷的可行性。本論文的主要目標在於結合以含兩性離子載體毛細管膠體 (CACG) 為介質的等電焦集電泳（IEF）與十二烷基磺酸鈉-聚丙烯醯胺膠體電泳（SDS-PAGE），開發出廉價而高效的二維電泳技術應用於唾液內蛋白質的分析，並做為人類疾病的早期檢測或蛋白質體學研究上的一種模式。
收集外表健康個體的唾液共十一件，其中非抽菸個體男女各三人，抽菸個體中男性兩人女性三人，後者於抽菸前、抽菸後及抽菸後30分鐘分別採集，檢體的收集採非刺激、自然流出方式。新鮮檢體經由離心處理後與含有尿素-兩性離子（urea-ampholyte）的單體溶液混合，再將此混合物置於冰上作超音波震盪處理，進而在室溫下抽氣15分鐘後於毛細管中凝膠。第一維 (IEF) 的分析使用含兩性離子載體毛細管膠體 (CACG) 電泳，接著使用12.5%十二烷基磺酸鈉-聚丙烯醯胺膠體電泳（SDS-PAGE）進行第二維的電泳分析，膠體以硝酸銀染色。蛋白質圖譜經掃描後以Image-Master軟體進行比對，再以質譜儀做蛋白質序列分析，以確認所屬蛋白質。
實驗結果由電泳圖譜顯示，唾液在低溫下蛋白質會產生沉澱導致在電泳過程中出現無法聚集的現象，可藉由上述步驟加以預防。使用此程序，可以在8小時內分析12-16檢體，通常只需10-20μl的唾液就可以進行分析，且花費較使用固定化酸鹼梯度條片 (IPGS) 進行IEF相對的低廉釵h。
由不同唾液檢體的分析結果顯示，非抽菸個體 (包括不同性別) 唾液的蛋白質圖譜並沒有明顯的差異。而抽菸和非抽菸者唾液的蛋白質圖譜則有明顯的差異；抽菸者比非抽菸者唾液內表現量上升的蛋白質是truncated?amylases，而cystatin SNs、parotid secretary proteins（PSP）及Zinc?2 glycoprotein表現量卻是呈現下降的趨勢。抽菸個體，不論男女，由抽菸前與抽菸後唾液的蛋白質圖譜做比較顯示：truncated ?amylases在抽菸後表現量會下降，但在抽菸30分鐘後就會回升；相反的是，Cystatin SNs在抽菸後表現量會上升，但在抽菸30分鐘後就逐漸回降。
Human saliva contains a large number of proteins and saliva sampling is a non-invasive process that can be easily obtained and are of great potential in clinical and epidemiological research. In this study, by a combination of IEF electrophoresis on carrier-ampholyte capillary gel (CACG) medium and SDS-PAGE, a cheap and high-performance mini-gel two-dimensional electrophoresis（2-DE）for salivary protein analysis was developed and used as a model in earlier diagnosis of diseases and proteomic study.
Totally 11 apparently healthy subjects, including 3 non-smoking male, 3 non-smoking female, 2 male smokers and 3 female smokers, were joined in this experiment. Saliva samples of each smoker were collected before, just after and after 30 minutes of smoking. Whole Saliva was collected by un-stimulated draining method.
A centrifuged fresh whole saliva sample was first mixed with urea-ampholytes-containing monomer solution; the mixture was sonicated in ice and incubated under vacuum for 15 min at room temperature. The CACG-IEF electrophoresis was carried out in the first dimension, and then followed by a 12.5% mini-SDS-PAGE. After the gel being stained was silver nitrate, the protein pattern on gel was scanned and further analyzed by software of Image Master from ImageMaster from A & B. Selected protein spots were excised, and examined by mass spectrometry.
It was found that degradation of salivary protein sample, and the protein aggregation formed under cold-storage of saliva, which might induce precipitation and non-focusing during the electrophoretic process, could most be prevented by the newly developed protocol. Using this procedure, as many as twelve to sixteen samples could be obtained in less than 8 hours. In general, only as low as 10-20µl of saliva was needed for each gel, and the cost was quite low as compared to IPGS-IEF.
The results showed that there was no significant difference between the non-smoker’s saliva samples of different gender. However, the saliva of smoker showed a different pattern from non-smoker; among them, truncated ?amylases were increased, but cyctatin SNs, parotid secretary proteins and Zn-? glycoproteins were decreased. The 2-DE pattern revealed that less amounts of truncated ?amylases in smoker’s saliva just after smoking, but it is gradually recovered within 30 minutes. On the other hand, cyctatin SNs in smoker’s saliva showed the reverse situation. |
