臺灣博碩士論文加值系統

乳癌今日仍是全球婦女中最為常見與最高致死率的癌症，乳癌治療緩解後，會因轉移導致復發而致死。雖經多年之研究，今日仍對乳癌的轉移機制了解不多，導致無法有效治癒乳癌。 METCAM/MUC18是一種類免疫球蛋白的細胞黏附份子，能促進表皮細胞腫瘤的血管增生與轉移，經由許多年的研究，本研究團隊及另一研究團隊之前發現METCAM/MUC18之表達會促進乳癌腫瘤之形成及轉移，可是在METCAM/MUC18對促進乳癌惡化的機制上仍然了解不多，發現在裸鼠模型下，一個METCAM/MUC18高表現的SK-BR-3細胞株在癌症發展初期會有暫時性的抑制，所以本研究的目標在為了了解此現象是由於：clone之差異、或是劑量之差異、抑或是人工製造的產物。為此我們又挑選出不同METCAM/MUC18表現的抗G418的SK-BR-3 clones，用這些clone做離體細胞移動性、侵略性、及細胞群落形成之測試 ; 並做裸鼠動物模式之測試，為了減低人工製造的可能，減低注射量為每隻裸鼠0.5x106個細胞 ; 最後做一些細胞傳訊路徑的指標分析，像是抗凋亡、生長指標Bc1-2、促凋亡指標Bax、細胞增生指標PCNA、survival pathway指標 phospho-AKT/AKT, 血管增生指標VEGF及VEGF-R2及有氧醣分解代謝指標LDH-A，並做組織免疫化學分析及組織切片。我們發現：METCAM/MUC18促進細胞移動及侵略性。METCAM/MUC18在動物模式中，其表現劑量與腫瘤發展成正比，由此得知從前觀察到的暫時性抑制，是一種由於注射過多細胞之人工製造之結果，而非由於clone之差異性，也非由於METCAM/MUC18劑量之差異。也知METCAM/MUC18表現促進腫瘤增生之機制可能是與survival pathway及血管新生有正相關。

Breast cancer is still the most prevalent and deadly cancer in women worldwide. The death reason of recurrent breast cancer is due to distant metastasis even after treatment. After many years of extensive studies, the mechanism and biology of breast cancer tumor formation and metastasis is still poorly understood. METCAM/MUC18, an Ig-like cell adhesion molecule, plays an important role in promoting angiogenesis, tumorigenesis and metastasis in several epithelial tumors. Over-expression of METCAM/MUC18, an Ig-like cell adhesion molecule, promotes tumorigenesis and progression of human breast cancer cells. We also observed an intriguing phenomenon that a high-expressing SK-BR-3 clone manifested a transient tumor suppression effect in vivo, but not another clone. The purpose of this study is to understand if this is due to the effect of clonal variation, dosage, or simply an artifact. Several G418-resistant clones of SK-BR-3, expressing different levels of METCAM/MUC18, were obtained for testing effects of METCAM/MUC18 on their in vitro behaviors and in vivo tumorigenesis in female athymic nude mice. Tumor sections were made for histology and immunohistochemistry (IHC) analyses and tumor lysates for Western blot analysis to determine effects of METCAM/MUC18 expression on levels of various downstream effectors. We observed that over-expression of METCAM/MUC18 promoted in vitro motility and invasiveness. The extent of in vitro tumorigenesis of SK-BR-3 cells was directly proportional to the dosage of the protein. Over-expression of METCAM/MUC18 promoted tumorigenesis of SK-BR-3 cells even when one tenth of cell number (0.5 x 106) was injected. Furthermore, the extent of in vivo tumorigenesis of SK-BR-3 cells was directly proportional to the dosage of the protein. Previously observed transient tumor suppression effect from the same clone was no longer observed. Thus we concluded that the transient suppression effect from the clone was not due to clonal variation or dosage effect, but rather due to an artifact created by injecting a high cell number (5 x 106). Downstream effectors, such as phospho-AKT/AKT ratios, VEGF and VEGF-R2, were elevated in the tumors. Thus, METCAM/MUC18 positively promotes tumorigenesis of SK-BR-3 cells via similar mechanism by increasing proliferation via augmenting survival pathway and angiogenesis.

目錄

摘要 i
Abstract iii
致謝辭 v
目錄 vi
圖表目錄 ix
第一章 緒論 1
1.1 臺灣乳癌簡介 1
1.2 癌症轉移 (Metastasis) 2
1.3 METCAM/MUC18簡介 2
1.4 METCAM/MUC18在乳癌上的角色 4
第二章 材料與方法 9
2.1 實驗藥品 9
2.2 溶液配置 13
2.3 細胞培養 16
2.3.1 細胞活化 17
2.3.2 培養基更換 17
2.3.3 繼代生長 18
2.3.4 冷凍細胞 18
2.4 蛋白質之定量及測定 19
2.4.1 細胞溶解、收集 19
2.4.2 蛋白質定量 20
2.4.3 十二硫酸脂鈉-多聚丙烯醯胺膠體電泳分析（Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE）20
2.4.4 西方點墨法 (Western Blot) 21
2.4.5 膠體染色、退染、保存 24
2.5 離體細胞測試 (In Vitro Cell Assay) 24
2.5.1 細胞移動性測試 (Cell Motility Assay) 24
2.5.2 細胞侵略性測試 (Cell Invasiveness Assay) 26
2.5.3 離體細胞群落形成測試 (In Vitro Colony Formation Assay) 26
2.6 動物模式 27
2.6.1 實驗動物飼育 27
2.6.2 腫瘤取出與保存 27
2.6.3 腫瘤蛋白萃取 28
2.6.4 免疫組織染色 28
2.7 統計分析 30
第三章 實驗結果 31
3.1 G418挑選出之SK-BR-3 clone之METCAM/MUC18表現量 31
3.2 METCAM/MUC18對離體人類乳癌細胞株侵略性與移動的影響 33
3.3 METCAM/MUC18表現量與In Vitro Colony Formation的關聯性 37
3.4 在低劑量注射下(0.5x106)SK-BR-3細胞株之METCAM/MUC18表現量越高越增加在雌性裸鼠之致瘤性及最終之腫瘤平均重量 39
3.5 在較高劑量注射下(2x106)SK-BR-3細胞株之METCAM/MUC18表現量與雌性裸鼠致瘤性與最終腫瘤重量，反而似乎無顯著差異 44
3.6 源自於SK-BR-3細胞株的腫瘤METCAM/MUC18的表現 51
3.7 SK-BR-3 clone之METCAM/MUC18所引起之致瘤性之初步機制研究 53
第四章 結論 60
第五章 未來展望 63
參考文獻 64

圖表目錄

圖一 METCAM/MUC18的結構 4
圖二 四種人類乳癌細胞株的METCAM/MUC18表現量 6
圖三 METCAM/MUC18表現對SK-BR-3乳癌細胞株腫瘤發生的影響 8
表一 西方點墨法所使用之抗體 22
圖四 利用西方點墨法分析每個clone/cell line的METCAM/MUC18表現量 32
圖五 METCAM/MUC18表現量對於細胞移動性的影響 35
圖六 METCAM/MUC18表現量對於細胞侵略性的影響 36
圖七 METCAM/MUC18表現對In Vitro Colony Formation的影響 38
圖八 在低劑量注射下(0.5x106)，METCAM/MUC18表現對雌性裸鼠之SK-BR-3 clone致瘤性、腫瘤發生率和最終平均腫瘤重量的影響 42
圖九 在高劑量注射下(2x106)，METCAM/MUC18表現對雌性裸鼠之SK-BR-3 clone致瘤性、腫瘤發生率和最終平均腫瘤重量的影響 49
圖十 SK-BR-3腫瘤的METCAM/MUC18的表現量與組織切片與組織免疫化學分析結果 52
圖十一 各項細胞傳訊路徑指標在SK-BR-3腫瘤上的表現 55
圖十二 METCAM/MUC18可能的傳訊路徑(預測) 62
圖十三 METCAM/MUC18影響AKT磷酸化而促進細胞存活、增生、移動性、侵略性、血管新生 62

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