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標題: 以銀增強奈米金標定大鼠、犬和雞之內質網酵素
Silver Enhanced Immunogold Localization on Endoplasmic Reticulum Enzymes of Rat, Dog and Chicken
作者: 陳彥伯
Chen, Yan-Bo
關鍵字: 銀增強奈米金
silver enhanced immunogold
endoplasmic reticulum
enzyme histochemistry
NADPH P450 reductase
出版社: 獸醫病理生物學研究所
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摘要: 傳統上細胞中內質網的結構,必須進行超薄切片,藉由穿透式電子顯微鏡以適當倍率做病理診斷和研究觀察,然而電顯製作過程相對於其他診斷技術較為困難。本研究採取大鼠、犬以及蛋雞正常無病變之肝臟、胰臟做為陽性以及脾臟做為陰性對照,以酵素組織化學與銀增強免疫金標定法之冷凍切片,標定內質網上特定酵素活性和專一性酵素蛋白質,簡化電顯切片製作的繁複過程,縮短判斷目標細胞中內質網之操作時間,達到簡化病理診斷之方法學改良。 本實驗分別以內質網膜上兩種蛋白NADPH-Cytochrome P450 Reductase與Glucose-6-Phosphatase之酵素染色法與免疫標定法進行染色,後以內質網之另一蛋白質Calreticulin對非催化蛋白做免疫標定。結果顯示NADPH-Cytochrome P450 Reductase與Glucose-6-Phosphatase酵素法,在大鼠、狗、蛋雞肝細胞以及脾臟邊緣區部分細胞有較強的訊號;而以免疫法對NADPH-Cytochrome P450 Reductase與Glucose-6-Phosphatase進行染色,在大鼠、狗肝細胞、非胰外分泌細胞與脾臟邊緣區細胞有較強的訊號。以免疫電子顯微鏡針對脾臟邊緣區細胞進行觀察,可於脾臟觀察巨噬細胞之內質網上有與銀增強免疫金標之訊號,確定邊緣區細胞為巨噬細胞。 根據染色結果有以下結論,免疫法較酵素組織化學法有更佳之敏感性與種別專一性,然而酵素法雖無敏感性較差,但較無種別限制性。各種細胞的標定蛋白質表現多寡為同時影響酵素法與免疫法之重要因素。透過免疫組織化學法標定內質網的數量,有潛力改善電顯於腫瘤及其他疾病之診斷。
Transmission electron microscopy (TEM) is widely utilized to tumor diagnosis through estimating the content of intracellular organelles in ultrastructural observation. However, the procedure of TEM is quite complicated comparing with general morphological technique. The target of this study was refinement of conventional TEM of endoplasmic reticulum (ER) identification and to constitute the basic data in localization of liver, pancreas and spleen. In this study, the methods enzyme histochemistry and silver enhanced nanogold immunolabeling to localize the NADPH-Cytochrome P450 reductase and Glucose-6-Phosphatase on ER membrane of rat, dog and chicken were used to replace the conventional immunoelectron microscopy. The immunolabeling was confirmed by the anti-calreticulin, which localized on ER, nuclear envelope and nucleus. The results of enzyme histochemistry stain manifested strong signals in the liver of rat and chicken, while the results of immunolabeling stain manifested the strong signals in the liver of rat and dog. In addition, the cell in the marginal zone of spleen displayed high intense stain in both enzyme and immune labeling. In order to recognize the positive stained cells in spleen, the immunoelectron microscopy was performed and the results revealed that the signals were ER of macrophage. In conclusion, the immune method displayed a better sensitivity and less cross-reaction than the enzyme method. Moreover, the protein expression in various tissues determined the outcome of the stain. Silver enhanced immunogold method was available to improve the tumor or disease diagnosis in veterinary medicine through the appropriate selection of target protein to localize the intracellular organelle.
其他識別: U0005-1707201323524600
Appears in Collections:獸醫病理生物學所



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