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標題: 犬血管肉瘤之KIT蛋白過度表現及c-kit基因缺失
KIT overexpression and a deletion in c-kit gene of canine hemangiosarcomas
作者: 陳易辰
Chen, Yi-Chen
關鍵字: c-kit基因
c-kit gene

出版社: 獸醫學系暨研究所
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摘要: 犬血管肉瘤是一種源自血管內皮細胞且極具侵犯性之惡性腫瘤,具高度轉移率及造成患犬高度死亡率的特性。KIT蛋白是屬於受體型酪胺酸激酶(receptor tyrosine kinase, RTK)中第三家族的一種跨膜蛋白,,具有調節許多種類細胞內訊息傳遞的功能。KIT蛋白的過度表現在許多種人類的肉瘤及動物的腫瘤中都有被偵測到。c-kit基因的突變會導致KIT蛋白不需與幹細胞因子(stem cell factor, SCF)結合,就可自行持續磷酸化而活化啟動下游的訊息傳遞。目前犬血管肉瘤的致病因子仍不清楚。本研究的主要目的包括(一)利用免疫組織化學染色的方式去偵測KIT蛋白在犬血管瘤及血管肉瘤中的表現情形及(二)從犬血管肉瘤組織抽取腫瘤細胞的RNA後添加反轉錄酶將其反轉錄成cDNA,再利用聚合酶鏈鎖反應(polymerase chain reaction, PCR) 及TA cloning技術將c-kit基因的開放閱讀框架(open reading frame, ORF)片段增幅後定序。研究樣本包含16個犬血管瘤和24個犬血管肉瘤的組織切片以及從7個犬血管肉瘤組織的腫瘤細胞萃取而得的cDNA。結果顯示KIT蛋白在犬血管肉瘤中的陽性表現率高達83.3% (20/24),而在全部的犬血管瘤中(16/16, 100%)皆未被偵測到(χ2 = 26.667, P < 0.0001)。在本研究所有犬血管肉瘤的RNA中(7/7, 100%),其c-kit基因序列所轉譯而成之胺基酸序列中都可發現在第513至516個密碼子之間有連續四個胺基酸序列發生缺失的現象。因此KIT蛋白過度表現可以用來區別血管來源之腫瘤的良惡性,而c-kit基因的缺失在犬血管肉瘤的致病機制中扮演著何種角色值得再做更進一步的研究與探討。
Canine hemangiosarcoma (HSA) is an aggressive malignant neoplasm of vascular endothelial cell origin and is characterized by a highly metastatic rate and fatality rate. KIT (CD117) is a transmembrane protein that belongs to the type III subfamily of the receptor tyrosine kinases operating in cell signal transduction of several cell types. KIT overpression has been variably reported in some human sarcomas and domestic animal tumors. c-kit gene activating mutations typically confer constitutional KIT phosphorylation and downstream activation independent of ligand binding. The aims of this study were to investigate the expression levels of KIT protein by immunohistochemical staining, and the cDNA of the open reading frame (ORF) encoding c-kit gene amplified by polymerase chain reaction and TA cloning followed by sequencing to demonstrate the mutation of c-kit gene in canine HSAs. Forty specimens including 16 canine hemangiomas (HAs) by surgical removal, 15 canine HSAs by surgical excision and 9 cutaneous canine HSAs by needle core biopsy, either frozen or paraffin-embedded tissues, were enrolled in this study. Totally, the cDNA obtained from 7 KIT-overexpression HSAs were evaluated for c-kit ORF. Results showed that the positive immunolabelling for KIT was detected in 83.3% of (20/24) HSAs and none of HAs (16/16, 100%) demonstrated immunoactivities of KIT (Chi-square: 26.667, P < 0.0001). Diffuse cytoplasmic staining pattern of neoplastic cells were observed in all KIT positive HSAs (20/20, 100%). A deletion, named as G513-K516del, composing of four amino acids translated by codons 513-516 located in exon 9 of the c-kit gene was detected in all selected HSAs (7/7, 100%). Therefore, it was suggested that overexpression of KIT can be used to distinguish benign or malignant vascular endothelial originated tumor, and the role of a deletion in c-kit gene in the pathogenesis of canine HSAs is worthy of further investigations.
其他識別: U0005-1607201210140300
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