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研究生:蘇厚有
研究生(外文):Hou-You Su
論文名稱:犬之乳腺腫瘤細胞株的建立與特徵分析
論文名稱(外文):Establishment and characterization of a cell line from canine mammary gland tumor
指導教授:王孟亮張仕杰
指導教授(外文):Min-Liang WongShih-Chieh Chang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:70
中文關鍵詞:犬之乳腺腫瘤細胞株肌上皮細胞犬之惡性混合型乳腺瘤裸鼠分化細胞骨架蛋白上皮表面膜蛋白
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乳房腫瘤在母犬為常見的腫瘤,依據組織學的分類,良性和惡性腫瘤的比例約各佔一半,其中又以混合型乳腺瘤最常發生。由於乳腺組織是由一群不同型態細胞所組成,因而構成複雜的組織學型態和病理變化,而增加試管內研究的複雜度,因此自犬乳腺腫瘤分離建立的細胞株,可提供作為腫瘤細胞增殖的機轉和發展抗腫瘤策略的研究。
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.
第一章 緒言………………………………………………………………1
第二章 文獻探討…………………………………………………………………2
第一節 犬乳腺腫瘤之探討……………………………………………………2
1-1 犬乳腺腫瘤流行病學調查………………………………………….2
1-2 臨床表現…………………………………………………………….2
1-3 組織病理學………………………………………………………….3
1-4 致病原理…………………………………………………………….3
第二節 乳腺腫瘤細胞株的建立與特質的分析………………………………5
2-1 乳腺腫瘤細胞株的建立…………………………………………...5
2-2 細胞型態學的研究………………………………………………...5
2-3 胚層來源…………………………………………………………...6
2-4 試管內腫瘤細胞培養的特性……………………………………...6
2-5 生物體內的試驗…………………………………………………...7
第三節 乳腺腫瘤細胞株的研究與應用………………………………………7
第三章 材料與方法………………………………………………………………..10
第一節 犬乳腺腫瘤組織的採樣……………………………………………..10
1-1 乳腺腫瘤患犬基本資料…………………………………………...10
1-2 外科治療…………………………………………………………...10
1-3 腫瘤組織的處理…………………………………………………...10
1-4 組織切片的製作…………………………………………………...11
第二節 乳腺腫瘤單一細胞的分離與初代培養……………………………..11
2-1 單一細胞的分離…………………………………………………..11
2-2 乳腺腫瘤細胞初代培養…………………………………………..11
2-3 細胞換液…………………………………………………………..12
第三節 細胞株的維持與保存………………………………………………..12
3-1 細胞第一次繼代…………………………..…………………….12
3-2.1 細胞連續繼代與維持……………………………….…………..12
3-2.2 初代細胞的冷凍保存………………………………………….. 13
第四節 購買的犬乳腺腫瘤細胞株的培養…………………………………..13
4-1 基本資料…………………………………………………………13
4-2.1 ATCC cell line 的培養與繼代………………………………….15
4-2.2 ATCC cell line的冷凍保存……………………………………..15
4-2.3 細胞的解凍……………………………………………………...15
第五節 細胞型態學分析……………………………………………………..16
5-1.1 接種細胞株至玻片的細胞培養盤……………………………..16
5-1.2 細胞的固定……………………………………………………..16
5-1.3 免疫細胞化學染色(1) …………………………………………16
5-2.1 接種細胞株至玻片的細胞培養盤……………….…………….17
5-2.2 細胞的固定……………………………………………………..18
5-2.3 免疫細胞化學染色(2)……………………………………...…..18
第六節 不同荷爾蒙藥物對細胞株腫瘤基因RNA表現的影響……………18
6-1.1 荷爾蒙藥物的配置……………………………………………..18
6-1.2 加入荷爾蒙藥物至細胞培養液中…………………………….18
6-1.3 Total RNA的抽取……………………………………………...19
6-1.4 RNA 膠體電泳………………………………………………..19
6-1.5 Transfer of RNA to nylon membrane………………………….20
6-1.6 Prehybridization………………………………………………..20
6-2 DNA Probe的製備……………………………………………..20
6-2.1 質體DNA的抽取……………………………………………..20
6-2.2 質體DNA酵素切割與insert DNA 的確認………………….21
6-2.3 Insert DNA的大量製備與純化……………………………….22
6-2.4 Isotope labeling of insert DNA………………………………...24
6-3 Hybridization …………………………………………………..25
6-4.1 Removal of probes from northern blots………………………..26
6-4.2 Reuse of blot…………………………………………………...26
第七節 細胞增殖速率(MTT proliferation test, Roche)……………………...26
第八節 Anchorage-independent growth test…………………………………27
第九節 BRCA2 gene sequence analysis……………………………………...27
9-1 細胞DNA的萃取 (Viogeneâ kit)………………………………27
9-2 Polymerase chain reaction (PCR)………………………………..28
9-3 Ligation 與E.coli的表現………………………………………29
9-4 核酸序列的分析…………………………………………………30
9-4.1 去除primer ……………………………………………………..30
9-4.2 加dye後的PCR sequencing反應………………………………30
第十節 Tumorigenicity in vivo……………………………………………….31
10.1 接種DMGT cell line至SCID mice……………………………31
10.2 接種DMGT cell line至nude mice. ……………………………32
10.3 老鼠身上腫塊的切除與切片的製作…………………………..32
第十一節 Karyotype analysis………………………………………………….32
第四章 結 果………………………………………………………………….32
第一節 犬之乳腺腫瘤組織病理學診斷…………………………………….33
第二節 細胞株光學顯微鏡的型態觀察…………………………………….33
第三節 細胞免疫化學染色分析…………………………………………….33
3-1 染色 (1)………………………………………………..…………33
3-2 染色 (2)………………………………………………………..…34
第四節 細胞增殖速率分析…………………………………………………..34
第五節 不同荷爾蒙藥物對細胞株腫瘤基因RNA表現的影響…………...35
第六節 BRCA2 gene exon 11 part sequence analysis………………………..35
第七節 Anchorage — independent growth…………………………………….35
第八節 Tumorigenicity in vivo……………………………………………….35
8-1 接種DMGT細胞株在SCID mice形成腫塊與組織切片型態….35
8-2 接種DMGT細胞株在nude mice形成腫塊與組織切片型態…..36第九節 Karyotype analysis………………………………………………….. 37
第五章 討 論………………………………………..…………………………53
附錄一 含GAPDH探針之質體結構……………………………………………..61
附錄二 含FAK探針之質體結構…………………………………………………62
附錄三 犬的BRCA2基因exon 11序列 (GenBank)…………….………………..63
參考文獻……………………………………………………………………………..65
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