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Establishment and characterization of a cell line from canine mammary gland tumor
實驗的進行是從本系獸醫教學醫院，採集患有乳腺腫瘤母犬的腫瘤組織，目前已成功的自一隻大於6歲、混種母犬的乳腺腫瘤組織分離出一株細胞 (命名為DMGT cell)。此一腫瘤組織經由病理切片診斷，結果為惡性混合型乳腺瘤。細胞在試管內培養的型態呈長條梭狀至多角形，在高密度的生長時，則形成多個聚集放射狀、細胞間互相交錯成網狀的生長方式。我們進一步以免疫細胞化學染色分析，此一細胞株對rabbit anti-human keratin、mouse anti-human mucin 1 antibodies呈陽性反應，而對mouse anti-vimentin antibody則為陰性反應，顯示這一建立的細胞株為上皮來源的細胞。細胞增殖速率試驗之結果顯示，DMGT cell的doubling time約為48小時。另外，為了瞭解DMGT cell是否有惡性腫瘤細胞的特性，將細胞培養在soft agar，兩週後可觀察到有cell colonies 的形成，並以MTT 確認其為有活性的細胞集結。在生物體內的試驗則將DMGT cell line接種至SCID mice的腹部皮下與裸鼠的flank region 皮下，大約一個月至一個半月之間，可觀察到mass的形成，將此腫塊切除後作病理切片的診斷，可見增生的組織形成多個islands的結構，細胞呈長條狀，細胞核呈圓形至橢圓形，並在一視野下可見多個有絲分裂期的細胞。由以上的結果，回應犬混合型乳腺腫瘤病理切片的型態，以及DMGT cell生長和型態學的分析，推測此細胞株為一有致腫瘤特性的乳腺肌上皮細胞。|
Mammary gland tumors are the most common types of neoplasms in bitch. According to histopathologic classification, the percentages of malignant tumors are about 50 %, and mixed type mammary gland tumors are usually developed. Because of the mammary gland is consisted of multiple cell types and forms a complex histogenesis and pathologic change, so that it is a difficult to research in vitro and vivo. If a cell line can be established from canine mammary gland tumor, it may be useful for the research of cell proliferation as well as for the development of anti-cancer reagent. Initially we obtained the mammary gland tumor from the 6 years old patient in the Veterinary Hospital of Chung-Hsing University, and isolated the cell from tumor. Histopathologic diagnosis of the tumor revealed a malignant mixed mammary gland tumor. We have successfully established a cell line, which was named DMGT. The DMGT cells were cultured in flask showing elongated spindle-shape and polygonal cell type. When they were grown to high density, the cell became multiple cluster, stellate-form, and arranged to cross-link. We further analyzed the properties of the cell line by immunocytochemistry. The immunocytochemistry staining results indicated the cells are keratin postive, mucin 1 postive, and vimentin negtive. So we concluded the cell line was a epithelial origin. In the MTT proliferation rate test, the doubling time of DMGT cells was about 48 h. Besides, to investigate the transforming characteristic of DMGT cells, we cultured them in soft agar. After two weeks, the cell focus was formed, and the MTT test demonstrated the cell colonies were viability. In malignancy test, we injected subcutaneously the DMGT cells into the abdomen of SCID mice and into the back of nude mice. About a month later, we observed the mass formation on inocultated site both in SCID mice and in nude mice. The mouse tumor masses were excised and histopathologic examination was performed. Islands of poor differentiated spindle-shaped cells were observed in SCID mouse tissue. Taken together, we established a transformed cell line (DMGT) from a canine mammary tumor, and the immunohistochemical property and the tumorigenecity of DMGT cells were studied.
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