研究於異種動物模式之具侵略性犬傳染性花柳性腫瘤的基因表現：鑑定應用於腫瘤惡性及轉移之臨床診斷標記

Genes associated with the aggressiveness of canine transmissible venereal tumor in a xenographic model: potential clinical application as indicators of malignancy and metastasis

雷漢容(Han-Jung Lei)

指導教授 ： 朱瑞民

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10546

摘要

為建立小鼠模式而將犬傳染性花柳性腫瘤（canine transmissible venereal tumor, CTVT）接種於免疫缺陷（NOD/SCID）小鼠（小鼠CTVT腫瘤簡稱為XCTVT）。 XCTVT 具有與CTVT相同之細胞學與組織學特性，亦表現出CTVT 之特異性基因片段LINE-c-myc。然而，將此小鼠CTVT腫瘤再次接種回犬隻後（以下簡稱為MCTVT），發現MCTVT的生長特性已有改變，它較CTVT 具有更快的生長速度、較多的mitotic figures、更大的腫瘤體積、及不易進入腫瘤消退期的特性。為了深入探討MCTVT 腫瘤轉趨惡性化的原因，我們使用Affymetrix microarray 分析MCTVT 與CTVT 腫瘤之基因表現狀況。兩倍分析的結果顯示，有136個基因在MCTVT表現量大幅上升，37個基因在MCTVT表現量大幅下降。其中許多基因與重要的生理調控因子，腫瘤發生因子，及免疫調節因子均有關。選擇在MCTVT表現量上升的30個基因，以即時定量聚合酶連鎖反應（Real-time RT-PCR）進行基因表現量的確認，發現有三個基因的表現量確實在MCTVT顯著的上升。此三基因分別為基質金屬蛋白酶-1（matrix metalloproteinase 1，MMP1）、載脂蛋白C1 （ApolipoproteinC-I，APOC1）及尿氨酸-3-單氧酶（kynurenine 3-monooxygenase，KMO）。為了解此三個基因與腫瘤惡性的相關性，我們進一步在犬乳房腫瘤以即時定量聚合酶連鎖反應測試表現量，最後發現KMO在良性腫瘤的表現量極低，然而在惡性腫瘤此基因表現量則顯著上升。此外在腫瘤發生轉移時會更顯著性的升高。更重要的是，當KMO上升時病犬的存活時間會大幅度縮短。本研究首次發現犬KMO表現量與腫瘤的惡性具正相關性，並與預後有關。因此本酵素可能成為一個診斷腫瘤惡性及轉移的標誌。

關鍵字

腫瘤標誌 ； 微陣列晶片 ； 尿氨酸-3-單氧酶犬傳染性花柳性腫瘤 ； 犬乳腺腫瘤

並列摘要

A xenograft NOD/SCID mouse model bearing canine transmissible venereal tumor (CTVT) (designated XCTVT) was created in this study. In addition to confirmation of the morphological similarities of the CTVT and XCTVT cells, insertion of the specific long interspersed nuclear element (LINE) gene fragment in the 5’ end of the c-myc gene characteristic of CTVT cells was also confirmed in XCTVT. However, when inoculated back into dogs (designated MCTVT), MCTVT exhibited much more aggressive growth and a much higher metastatic frequency than common CTVT. The goal of this research was to explore the differences in gene expression between dog and mouse CTVT and determine the genes involved in the aggressive growth. The differential gene expressions of the two tumors were analyzed using Affymetrix, and in total, 136 genes were identified as being significantly up-regulated and 37 significantly down-regulated in MCTVT in comparison with CTVT by hierarchical analysis and filtering of the differentially-expressed genes by the 2-fold method. Many of these genes are involved in activities such as malignant candidate genes, important biological processes, tumor development and immune modulation, as has been reported previously. Three genes (APOC1, MMP1 and KMO) were highly up-regulated as confirmed by real-time RT-PCR and were tested in 35 spontaneous canine mammary gland tumors (MGT). The real-time RT-PCR results indicated that KMO and MMP1 discriminated malignant from benign MGT, and in addition, KMO gene expression was significantly higher in stages 4 and 5 malignant MGT than MGT of lower stages. The results of KMO mRNA level and IDO mRNA/protein data indicated that KMO up-regulation was independent of IDO. Over-expression of KMO was also found to be associated with poor prognosis in MGT dogs. In conclusion, KMO expression may have the potential to be used as a biological indicator to differentiate degrees of malignancy.

並列關鍵字

Biomarker ； Microarray ； CTVT ； Mammary gland tumor ； kynurenine 3-monooxygenase

參考文獻

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