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研究生:李恩惠
研究生(外文):En-Hui Lee
論文名稱:分離和辨認高轉移率鼻咽癌細胞
論文名稱(外文):Isolation and Molecular Characterization of Highly Metastasizing Nasopharyngeal Carcinoma Cells
指導教授:林榮寵
指導教授(外文):Jung-Chung Lin
學位類別:碩士
校院名稱:慈濟大學
系所名稱:微免暨分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:96
語文別:英文
論文頁數:51
中文關鍵詞:高轉移率鼻咽癌
外文關鍵詞:Metastasizing Nasopharyngeal Carcinoma cells
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化學療法和放射療法是兩種有效治療鼻咽癌的方法,然而,癌細胞的遠距離轉移卻往往造成治療上的失敗。 我們從鼻咽癌患者分離出轉移到骨髓的鼻咽癌上皮細胞株 (NPC-BM1) 中進一步再分離出不轉移的NPC-BM00,以及高轉移率的NPC-BM29 二種細胞株。 其中NPC-BM29 受到上皮間質轉化 (EMT) 的影響表現出侵入性,由上皮類型轉分化為間質顯型。 NPC-BM00 則表現出典型的多邊形細胞型態,而 NPC-BM29 則可能以各種型態表現,舉凡單極的折射紡錘狀,以及雙極的細胞質突起,或是細長的紡錘狀等。 在 2% 的血清中,NPC-BM29 細胞分裂一次的平均時間比 NPC-BM00 要來得快,分別是 26.76 小時和 30.67 小時。 從創傷-癒合之細胞移動 (Scrape-wound Migration) 實驗中發現不管腫瘤促進因子 (TPA),甘草酸 (GL),和抗腫瘤藥物 (HPMPC) 的有無,NPC-BM29 的能動性都比 NPC-BM00 還要高。 轉移的潛在性可以由 92-kDa typeIV collagenases/gelatinase B (MMP-2 和 MMP-9) 的差異性基因表現得知;兩種酵素都和腫瘤細胞的入侵和轉移有關。 NPC-BM29細胞表現MMP-9比NPC-BM00細胞大約高10倍。 與 NPC-BM00 相比較,NPC-BM29 的侵入表現型伴隨著抑制 E-cadherin 以及 β-catenin 的表現,以及提高 Ets1 轉錄因子的表現率。 由流式細胞分析對細胞表面標誌和腫瘤相關分子的 immunophenotypic characterization 可以看出,在 NPC-BM29 中AE3,MAK6 (細胞角質蛋白 8,14,15,16,18和19 的混合),E-cadherin以及 sialyl Tn 的產生非常明顯,而在 NPC-BM00 中則是 CEA 和 CD54 產生率有顯著的提高。 在 cytokine array ELISA 中,我們發現在第一天 NPC-BM29 相對於 NPC-BM00 來說釋放極高量的 IL-6,IL-8,IL-10 和 IL-13,而兩者在 IL-2 的釋放率中並沒有什麼差別。 值得注意的是,到了第三天,NPC-BM00 製造的 IL-6 比 NPC-BM29 還要多出三倍,然而 IL-8,IL-10和 IL-13 的量與第一天相去無異。 到了第五天,兩者皆保持著差不多高的 IL-6 產生率。 儘管 NPC-BM29 在 IL-6 的產生率並不像 NPC-BM00 一樣,整個試驗中卻持續著產生較高量的 IL-8 和 IL-10。 總括來說,在這項研究中,由上皮形變為間質的特徵以及與這些改變相關的潛在分子,提供了針對研發抗腫瘤細胞轉移的藥物一個舉足輕重的線索。
Concurrent chemotherapy and radiotherapy are effective in treatment of nasopharyngeal carcinoma (NPC). Distant metastasis, however, is the major cause of failure. NPC-BM1, an epithelial cell line established from a bone marrow biopsy of NPC patient, was further separated into non-metastasizing and highly metastasizing cell clones, designated NPC-BM00 and NPC-BM29, respectively. The metastasizing clone NPC-BM29 acquired the invasive phenotype marked by epithelial mesenchymal transition (EMT), transdifferentiation from epithelial type to mesenchymal phenotype. NPC-BM00 cells exhibited typical well-attached polygonal epithelial cell morphology. In contrast, NPC-BM29 was heterogeneous, from refractile, spindle-shaped cells with monopolar and occasional bipolar cytoplasmic processes, to elongated fibroblast-like shape cells. NPC-BM29 cells grew better in 2% serum than the non-metastasizing NPC-BM00 cells, with population doubling time of 26.76 h and 30.67 h, respectively. The scrape-wound migration assay revealed that NPC-BM29 cells exhibit higher motility than NPC-BM00, both in the presence and absence of 12-O-tetradecanyol-phorbol-13-acetate (TPA), glycyrrhizic acid (GL) and HPMPC (Cidofovir). The metastasis potential was confirmed by differential expression of 92-kDa type IV collagenases/gelatinase B (MMP-2 and MMP-9); both enzymes are involved in tumor cell invasion and metastasis. NPC-BM29 cells express approximately more than 10-fold of MMP-9 than NPC-BM00 cells. Compared to NPC-BM00, the acquisition of the invasive phenotype in NPC-BM29 was accompanied by downregulation of E-cadherin and its associated protein�n��-catenin, and with concomitant increased the expression of the transcription factor Ets1. Immunophenotypic characterization of cell surface markers and tumor-associated molecules by Flow cytometric analysis indicated that AE3, MAK6 (a mixture of cytokeratins 8, 14, 15, 16, 18, and 19), E-cadherin and sialyl Tn were significantly upregulated in NPC-BM29 cells, whereas the expression of CEA and CD54 were significantly increased in NPC-BM00 cells. In cytokine array ELISA, we found that
NPC-BM29 consistently released higher levels of IL-6, IL-8, IL-10, and IL-13 than that released by NPC-BM00 cells on day 1. There was no difference in the expression level of IL-2a in both cell lines. Interestingly, on day 3 the production of IL-6 by NPC-BM00 was markedly increased to approximately 3-fold higher than that of released by NPC-BM29. However, the production of IL-8, IL-10, and IL-13 on day 3 remained at the similar level as on day 1. On day 5, both cell lines maintained similar high levels of IL-6 production. Despite the fluctuation of expression level of IL-6, NPC-BM29 consistently produced higher levels of IL-8 and IL-10 throughout the entire period of assays. Taken together, the characterization of morphologic changes from epithelial to mesenchymal transition and the identification of the underlying molecules associated with these changes described in this study provide a lead for the design of specific anti-invasive drugs.
Contents…………………………………………………1
致謝……………………………………………………..2
Abstract ………………………………………………...3-4
中文摘要.….……………………………………………5-6
Introduction …………………………………………….7-10
Material & Methods………………………………...…..11-16
Results…………………………………………….…….17-21
Discussion……………………………………………....22-27
Acknowledges………..…………………………………28
References………………………………………………29-33
Tables …………………………………………………..34
Legends to Figures….………………………………..…35-37
Figures …………………………………………………38-49
Appendix…………………………………………….…50-51
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