偵測犬惡性腫瘤中黑色素細胞抗原-A(MAGE-A)轉錄物與蛋白質的表現

Abstract

黑色素細胞瘤抗原-A (Melanoma antigen-A, MAGE-A)是一種主要廣泛表現於各種腫瘤中的抗原，然而，在正常組織中只表現在人類的胎盤和睪丸，並且高度表現MAGE-A抗原的病患相較於其它病患通常會有較差的預後。而MAGE-A在狗的表現情形，根據本實驗室先前的研究，到目前為止可知在狗正常組織MAGE-A蛋白質的表現只侷限在睪丸及卵細胞中 而多種間質來源的腫瘤中也可發現MAGE-A蛋白質的表現。本次實驗的主要目的是分別以免疫組織化學染色法及逆轉錄聚合酶連鎖反應偵測狗正常睪丸及多種惡性腫瘤中的MAGE-A蛋白質和轉錄物的表現。免疫組織化學染色法使用的是抗體是鼠抗人的單株抗體「6C1」，此抗體可成功偵測人類的MAGE-A1, -A2, -A3, -A4, -A6, -A10, -A12。聚合酶連鎖反應的引子則是由GenBank中狗MAGE-A相關基因序列的保留片段來進行設計。最終本實驗共有10個正常睪丸及44個惡性腫瘤組織進行分析。其中44個惡性腫瘤包含4個纖維組織肉瘤、5個脂肪肉瘤、3個惡性周邊神經鞘瘤、3個傳染性花柳性腫瘤、2個血管肉瘤、5個惡性黑色素瘤、4個淋巴瘤、1個組織細胞肉瘤、5個肥大細胞瘤、2個黏液肉瘤、4個骨肉瘤、3個漿細胞瘤和3個惡性鱗狀上皮細胞瘤。聚合酶連鎖反應的產物大小為379 bp，在本實驗中將此產物命名為MAGE-A379。免疫組織化學染色法及逆轉錄聚合酶連鎖反應的結果相比下可知MAGE-A379 RNA是確實存在於正常睪丸及惡性腫瘤中的。在正常睪丸中MAGE-A蛋白質及MAGE-A379表現率皆為100%。而在惡性腫瘤中MAGE-A379的表現率為88.6% (39/44)，是高於MAEG-A蛋白質的表現率。而在黑色素瘤、血管肉瘤、惡性周邊神經鞘瘤、傳染性花柳性腫瘤、纖維組織肉瘤、惡性黏液肉瘤和骨肉瘤中MAGE-A379的表現率為100%。將這39個惡性腫瘤中的MAGE-A379進行序列比對分析，其彼此的相似度為78.8%至100%。接著將這39條序列進行親源演化分析，可以發現在同一種腫瘤類別中其關係是較不同腫瘤來得接近的。根據此分析結果，我們推測在犬惡性腫瘤中的主要表現的MAGE-A亞型會因為腫瘤類型的不同而有所變化。總結來說，根據免疫組織化學染色法及逆轉錄聚合酶連鎖反應的結果，可知MAGE-A在犬正常睪丸及惡性腫瘤中具有穩定的表現，相信未來MAGE-A可能成為一個十分具有潛力的犬惡性腫瘤標記。

Melanoma antigen-A (MAGE-A) predominately expressed in tumors, whereas expression in normal tissue was limited to placentas and testes in human and the high expression of MAGE-A was also associated with poor outcome. Up to now, little is known about the expression of MAGE-A in dogs. Our previous study indicated that the expression of MAGE-A was restricted in canine testes and oocytes, and was predominately expressed in various types of mesenchymal tumors. The aim of this study is to detect the MAGE-A proteins and the MAGE-A transcripts in normal testes and various malignant tumors by immunohistochemistry (IHC) staining and reverse transcription polymerase chain reaction (RT-PCR), respectively. The primary antibody was mouse anti-human monoclonal antibody 6C1 against human MAGE-A1,-A2, -A3, -A4, -A6, -A10, -A12. A pair of primers was designed to target the conserved region of canine MAGE-A members. In total, 10 normal testes from dogs in varied ages and 44 tumors including 4 fibrosarcomas, 5 liposarcomas, 3 malignant peripheral nerves sheath tumors (MPNSTs), 3 transmissible venereal tumors (TVTs), 2 hemangiosarcomas (HSAs), 5 melanomas, 4 lymphomas, 1 histocytic sarcoma, 5 mast cell tumors, 2 myxosarcomas, 4 osteosarcomas, 3 plasmacytomas ,and 3 squamous cell carcinomas were analyzed. Given the size of product amplified by PCR was 379 bp, the amplicon was named MAGE-A379 herein. Consistent with IHC results, MAGE-A379 RNA was indeed expressed in most malignant tumors and normal canine testes. In normal testes, the positive rate of MAGE-A379 was 100% that was same as the result of IHC. In malignant tumors, the positive rate of MAGE-A379 was 88.6% (39/44), higher than that of IHC. Further, the expression of MAGE-A379 was 100% in melanomas, HSAs, MPNSTs, TVTs, fibrosarcomas, mxyosarcomas and osteosarcomas. Sequence alignment indicated that MAGE-A379 in malignant tumors shared identities ranging from 78.8% to 100%. Phylogenetic analysis of MAGE-A379 in malignant tumors indicated that the MAGE-A sequences were much closely related between the same tumor type than that between different tumor types. It was speculated that expression of the MAGE-A subtypes in malignant tumors might be varied with the different tumor types. Taken together, both IHC and RT-PCR showed MAGE-A was stable expressed in normal testes and malignant tumors. It might be a potential marker for canine malignant tumors.