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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/93005
標題: Detection of MAGE-A in Normal Canine Tissues by Reverse Transcription-Polymerase Chain Reaction
以反轉錄-聚合酶鏈鎖反應偵測犬正常組織的MAGE-A基因表現
作者: Yen-Hao Lai
賴彥豪
關鍵字: MAGE-A基因;犬;正常組織;反轉錄-聚合酶鏈鎖反應;聚合酶鏈鎖反應;MAGE-A;dogs;normal tissues;PCR;RT-PCR;real-time PCR
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摘要: 
在人類的各種惡性腫瘤中已證實有MAGE-A基因的表現，而體內的胞殺性T細胞會辨識此基因轉譯出之MAGE-A蛋白。除了成人睪丸、胎兒卵巢及胎盤，其餘正常組織皆不會表現MAGE-A基因，然而此基因在犬正常組織的表現特性仍未被研究。本實驗的目的為利用聚合酶鏈鎖反應(polymerase chain reaction)及即時定量聚合酶鏈鎖反應(quantitative real-time polymerase chain reaction)偵測犬正常組織中MAGE-A基因的表現並比較此二種偵測方法的一致性。研究樣本來自2隻幼年與3隻成年經標準程序之動物實驗核可後已施行安死術的健康犬，分別由5隻犬的不同器官分別取得39、42、40、43 及 33個，共197個正常組織，包括神經 (n=35)、消化道 (n=45)、免疫 (n=20)、呼吸 (n=9)、循環 (n=20)、內分泌腺體(n=18)、泌尿道(n=17)、生殖 (n=10)、肌肉骨骼 (n=13)與外皮 (n=10)等組織進行MAGE-A基因偵測。將組織樣本均質化後，萃取總核醣核酸(total RNA)進行反轉錄(reverse transcription)，以犬MAGE-A高度保留區(conserved region)設計之引子(primer)偵測基因表現。經由聚合酶鏈鎖反應，所有犬正常組織除了睪丸，其他組織皆未偵測到MAGE-A基因的表現。以敏感度更高的即時定量聚合酶鏈鎖反應進行偵測並量化MAGE-A與GAPDH之相對表現量後，發現睪丸中MAGE-A表現量為GAPDH的0.25倍；有少數樣本(n = 10/131)呈現MAGE-A陽性，其中6個樣本僅有微量相對表現量，其餘4個樣本皆來自於消化道系統，而多數的組織仍呈現MAGE-A陰性；在所有來自雄性犬的組織中，MAGE-A陰性樣本則占98% (n = 100/102)。比較兩種偵測MAGE-A基因的方式後，兩者的觀察值有高度的一致性，且陰性反應的偵測結果也近於完全一致。因此，經由兩種方法偵測可證實在犬正常組織中除了睪丸之外，MAGE-A基因的表現是有限的。

The MAGE-A, known as a cancer/testis antigen, expresses in various malignant tumors and normal testes in humans and can be recognized by cytotoxtic T lymphocytes. Since the expression of MAGE-A is unclear in normal canine tissues, the aim of this study is to detect MAGE-A by conventional and real-time polymerase chain reaction (PCR) to determine the expression of MAGE-A in normal canine tissues. Five clinically healthy dogs obtained from Taichung City Animal Protection and Health Inspection Office were approved by Institutional Animal Care and Use Committee and analyzed in the study. A total of 197 normal canine tissues from nervous (n=35), alimentary (n=45), immune (n=20), respiratory (n=9), circulatory (n=20), endocrine (n=18), urinary (n=17), reproductive (n=10), muscle-skeletal (n=13) and integumentary (n=10) organs were collected from these five dogs (39, 42, 40, 43 and 33 specimens, respectively). All cDNA were reverse transcribed from total RNA to detect MAGE-A expression by canine MAGE-A primers targeting canine MAGE-A conserved regions. Results demonstrated that MAGE-A expression in all of specimens except for testes were undetectable by end-point PCR. Among analyzed samples by real-time PCR, 10 specimens showed MAGE-A expression in addition to testes, while other tissues were negative on MAGE-A expression. Among them, 6 samples revealed weak expression of MAGE-A. However, there were no statistically significant difference between expression levels and tissue systems. Compare with these two methods of detecting gene, there was a high agreement of observed agreement and an almost perfect agreement of negative results. In conclusion, limitation of MAGE-A expression in normal canine tissues except for testes was confirmed.
URI: http://hdl.handle.net/11455/93005
其他識別: U0005-0608201511345500
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