臺灣博碩士論文加值系統

呼吸上皮纖毛形態的改變，可作為呼吸性毒性物質毒害程度的指標，但要取得沒有黏液層覆蓋的上皮標本相當困難。以掃描式電子顯微鏡觀察標本，除了掃描式電子顯微鏡本身之性能外，還要在標本製備上加以改進。
研究細胞或器官外表超微構造的先決條件，就是對標本作最適當的處理。從取樣到電子顯微鏡觀察的每一步驟，都必需極端的謹慎、正確和完善，方能獲得最佳的結果。在觀察呼吸上皮形態的研究中，對呼吸上皮的黏液層有各種不同的處理方式，標本未經沖洗處理、其組織表面黏液未加以清除則不能完整的呈現出呼吸上皮的超微構造，因此不易觀察。呼吸上皮掃描式電子顯微鏡標本製作的過程，經過固定、脫水、乾燥及鍍導電層等步驟，若操作不當、就無法觀察到標本之原貌。
本研究的目的是以磷酸緩衝溶液在低溫及連續震盪下清除呼吸道黏液並比較其清除的效果，以期定出有效的掃描式電子顯微鏡標本的製作流程。經由不同流程處理後發現，製作呼吸上皮的掃描式電子顯微鏡標本應先經由沖洗的步驟。而不同沖洗方法所得到的結果有極大的差異，比較不同的沖洗液後，發現以磷酸緩衝溶液處理可得到最理想的呼吸上皮的形態。故磷酸緩衝溶液最適宜作為掃描式電子顯微鏡標本製作過程中的沖洗液。由實驗結果可知，灌流固定後的呼吸上皮，先經磷酸緩衝溶液在低溫下沖洗，再分別經戊二醛－對甲醛、四氧化鋨固定，不同濃度的酒精脫水，液態二氧化碳臨界點乾燥後鍍金膜，可顯示出完整、清晰的纖毛及各種表面構造。

The morphological change of ciliated epithelium was applied as the indicator of toxic chemicals onto the respiratory system. Scanning electron microscopy（SEM）has been utilized to study the ultrastructural morphology of the respiratory tract such as the nasal cavities, larynx, trachea, principal bronchus, and lobar bronchus. The specimen surface observed under the SEM should be free of mucus or protein-rich body fluids, or other contamination. Thus, many techniques are developed to obtain better SEM specimens of the respiratory tract. The objective of this study is to develop a proper rinsing protocol for better SEM observation. Male rat respiratory tracts was taken and fixed with aldehyde in-situ and critical point dried. Many current treatment techniques were evaluated with the method developed in this study. It was found that with the repeated rinse of phosphate buffered solution followed by aldehyde fixation, the treatment will effectively eliminate the mucus coverage.

目次
中文摘要------------------------------------------------------------------------------------1
英文摘要------------------------------------------------------------------------------------2
第一章、前言------------------------------------------------------------------------------3
第二章、相關研究-----------------------------------------------------------------------4
一、 大白鼠的呼吸系統-----------------------------------------------------------4
二、 呼吸上皮掃描式電子顯微鏡標本製備研究的回顧----------------10
第三章、材料與方法-------------------------------------------------------------------17
一、 實驗流程--------------------------------------------------------------------17
二、 實驗動物--------------------------------------------------------------------17
三、 儀器設備--------------------------------------------------------------------17
四、 實驗藥品--------------------------------------------------------------------17
五、 實驗設計--------------------------------------------------------------------18
六、 大白鼠呼吸上皮掃描式電子顯微鏡標本製備步驟-------------19
第四章、結果與討論------------------------------------------------------------------22
一、 清除呼吸上皮的黏液層---------------------------------------------------22
二、 以掃描式電子顯微鏡觀察呼吸上皮的形態-------------------------22
三、 清洗的目的以及使用磷酸緩衝溶液沖洗標本
清除黏液層的原因---------------------------------------------------------38
第五章、結論---------------------------------------------------------------------------40
第六章、參考文獻---------------------------------------------------------------------44
第七章、附錄---------------------------------------------------------------------------47
表目錄
表4-1 呼吸上皮纖毛形態在不同清除黏液層步驟後的比較-----------------23
表5-1 掃描式電子顯微鏡標本製作流程的比較---------------------------------41
圖目錄
圖2-1 鼻腔（大白鼠頭部正中旁矢狀切面）-------------------------------------5
圖2-2 大白鼠支氣管樹的背面觀-----------------------------------------------------7
圖4-1 以磷酸緩衝溶液沖洗呼道不同部位之結果呈現-----------------------25
圖4-2 呼吸道不同部位未經沖洗步驟之結果呈現-----------------------------29
圖4-3 以生理鹽水沖洗呼吸道不同部位之結果呈現--------------------------32
圖4-4 標本以蛋白脢溶液泡浸4小時之結果呈現-----------------------------35
圖4-5 標本以蛋白脢溶液泡浸2小時之結果呈現-----------------------------37
圖5-1 掃描式電子顯微鏡用標本之製備法比較--------------------------------43

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