以傅利葉轉換式紅外線光譜法探討細胞及組織之蛋白質二級結構變
化
STUDY ON FT-IR MICROSCOPIC CHARACTERIZATION OF CELLS AND TISSUES IN PROTEIN SECONDARY STRUCTURE
中文摘要
目前對於癌化組織及細胞的檢測及其病理結果皆是以傳統的病理切片及利用顯 微鏡觀察其組織型態(Morphology)之判定；然而傳統的病理組織
(histopathological analysis)分析檢驗尚存在某些缺點，因此發展並建立一個 診斷新方法及技術並配合選定一個的標準模式、參數及檢測樣品製作的條件，並 由此模式及參數來獲得一種合理且新的癌症快速鑑別方式。本研究主要藉由冷凍 切片及傅利轉換紅外線光譜法，有系統地進行基礎資料收集與分析，再與傳統的 病理切片進行比對，以為未來的臨床診斷或治療上提供重要的參考依據。在組織 部分的實驗方法上，在取下組織後，以冷凍包埋液將適當大小的樣本包埋至於 -80OC 下存放，於-25OC 下進行冷凍切片厚度約 5mm，然後以
FT-IR/Microscope 觀察正常組織與惡化組織之蛋白質二級結構變化情形 ; 而 在細胞部分，則選擇具貼附性質之大腸癌cell line(HT29, Colo 205)及肝癌細 胞(Hep G2, Hep 3B)，使其貼附於已經前處理過之第二型鈦金屬圓片、選擇解
析度為4cm-1 為本研究所用，並以相同之方式觀察細胞樣本之可見光及紅外線
光譜資訊並以Deconvolution 及 Curve fitting 等方式進行波峰之分離並加以 分析統計。而就所得的結果而論，診斷為Carcinoma 中:β—sheet 由原來正 常組織的24.26％變為 20.05%，Random coil 由 25.50％變為 22.71％，α—
helix 由 29.54％變為 25.01％，β—turn 由 20.46％變為 32.08％；診斷為 Adenocarcinoma 中: β—sheet 由原來的 20.39％變為 20.19％，Random coil 由 24.71％變為 18.78％，α—helix 由 33.50％變為 20.91％，β—turn
由21.40％變為 40.08％癌化組織之β結構含量較正常組織之含量為高，亦即
波峰向高波數位移之傾向；細胞方面，則取決於加入酒精濃度與量的多寡，濃度
高者波峰有向低波數位移之傾向並且細胞有萎縮之情形，另外在Colo 205 的研
究中，β—sheet, Random coil, α—helix, β—turn 分別所代表在蛋白質二 級結構百分比為35.42 %, 28.01 %, 19.77 %, 16.79 %，可以得知 Colo 205 在 Amide I 中β—sheet 含量最高，加上β—turn 之含量，則屬於β型之 二級結構其含量將超過一半以上，恰可與大腸直腸癌組織之結果互為呼應。未來 希望對各種不同疾病與正常情況下進行比對，並找出其規則性進而作為將來臨床 診斷判讀的依據。
英文摘要
At present the approach to diagnosis malignant tissues and cells all are by
pathological section and use microscopy to observe their morphology. However, the traditional histopathological analysis still remaining in certain lacks and
Fourier-transform infrared spectroscopy (FT-IR) employs a unique approach to optical diagnosis of tissue pathology based on the characteristic molecular vibrational spectra of the tissue, because of these advantages it is a powerful tool for diagnosis in medical field. Therefore it is important for our study to establish a pattern and from this
pattern we can get novel and reasonable method for diagnosis of the cancer. By alternately compares with the changes of protein secondary structure in malignant and normal tissues and cells provides the deep rich insight the understanding to at present still not clear some cancer lines. The steps of this study (1) select the sample from malignant and normal tissues, (2) preparation and selection of the attachment (3) the FT-IR special absorption peak alternately compares (4) the changes of protein
secondary structure in cells which administrate drugs or not. It provides an important reference for clinical cure or diagnosis, with systemic data base collection and analyses through the methods of frozen cut slide and FT-IR spectroscopy.
In tissue, the spacemen embedding deposits as for -80OC for fresh-frozen, and prepare to 5mm thickness at -25OC for frozen -sectioning , then by FT-IR
spectroscopy observes the changes of protein secondary structure in malignant and normal tissues. In cell, colon cancer cell line (HT29, Colo 205) and hepatoma (Hep G2, Hep 3B) which with attach character are selected, then attach on the prepared Ti disc, wafer and glass disc, by same way to get the information from the optical and IR spectra. Then the peaks were deconvoluted by the Peak Fit software. The purpose in this study is to compare the alternation of protein secondary structure between α-helix and β-type conformations in amide I region. In the tissue, the percentage of β-type conformation in tumoral tissue is higher than it''s in normal tissue, and there is a trend that the peak shift to the higher wavenumber. The result in the cell depends on the concentration of the EtOH which were treated to the cancer cell. There is a trend that peak shift to lower wavenumber in higher concentration than in the lower concentration of EtOH. In the future, we hope that FT-IR can be used in different diseases and compare with the normal one. Further more, find the regular role to use the information in the diagnosis or cure in the clinic.
