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研究生： 洪曉琴
Hong, Hsiao-chin
論文名稱： Galectin-1會促進口腔腫瘤的形成與肌纖維母細胞的轉形分化
Galectin-1 promotes oral tumorigenesis and transdifferentiation of myofibroblasts
指導教授： 陳玉玲
Chen, Yuh-Ling
洪澤民
Hong, Tse-Ming
學位類別： 碩士
Master
系所名稱： 醫學院 - 口腔醫學研究所
Institute of Oral Medicine
論文出版年： 2009
畢業學年度： 97
語文別： 中文
論文頁數： 79
中文關鍵詞： 肌纖維母細胞
外文關鍵詞： myofibroblasts
相關次數： 點閱：45  下載：11 
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    • 腫瘤相關的纖維母細胞已經知道會調控多種固體瘤的產生與進展。之前的研究結果已經證明，Galectin-1高量表現在早期口腔癌的腫瘤相關纖維母細胞，其表現量與早期口腔癌的預後有正相關（p<0.05）。Galectin-1是一種碳水化合物結合的蛋白，由二個已知的次單元所組成，各自含有碳水化合辨識區。Galectin-1已被證實的生物功能包含：細胞凋亡、細胞轉形、細胞黏附、細胞移動的調控，甚至腫瘤轉移過程的影響。然而是否Galectin-1與口腔癌形成的過程，以及腫瘤相關的纖維母細胞的轉形分化有關，仍然未知。在實驗中我們利用Galectin-1基因剔除小鼠來分析是否缺少Galectin-1可能會減少4-Nitroquinoline-1-oxide誘發的口腔癌。在我們的結果中發現，wild type的老鼠不論是癌症前期病變或口腔癌，其發生率皆較高於Galectin-1基因剔除小鼠。另一方面，我們分離出健康的口腔牙齦纖維母細胞Human gingival fibroblasts（HGFs）與口腔腫瘤相關的纖維母細胞Cancer associated fibroblasts（CAFs），並且證明在CAFs內Galectin-1的表現會高於HGFs。利用類病毒short hairpin RNA (shRNA)載體去減少CAFs的Galectin-1的表現，會使肌纖維母細胞（myofibroblasts）的指標蛋白α-smooth muscle actin（SMA）的表現量也會減少。我們也發現到減少肌纖維母細胞的Galectin-1基因時，其狀態培養液（Condition media）誘導口腔癌細胞移行與侵襲的能力也會減少。在機轉的研究上發現，減少CAFs內Galectin-1表現的狀態培養液會減少蛋白質金屬酶2（MMP2），以及多種重要的細胞激素如單核球趨化蛋白-1（MCP-1）的表現，以MCP-1抗體中和肌纖維母細胞狀態培養液的MCP-1，則可有效抑制狀態培養液誘導的癌細胞移行。綜合以上結果顯示，Galectin-1與肌纖維母細胞的分化有關，並且在Galectin-1高度表現的CAFs會增加MMP2與細胞激素的產生，進而誘導口腔腫瘤細胞的移行與侵襲。這樣的發現顯示了 Galectin-1對於口腔鱗狀上皮細胞癌的形成有促進的作用。期望未來可以利用Galectin-1當作新的腫瘤預防或是治療策略的標的。

    Carcinoma-associated fibroblasts (CAFs) have been showed to regulate the initiation and progression of many types of solid tumors. Our previous results have been shown that the expression of Galectin-1 (Gal-1) is significantly up-regulated in the CAFs during early oral carcinogenesis (P < 0.05) and positively correlated with cancer prognosis. Gal-1 is a carbohydrate-binding protein composed of two identical subunits, each one containing a carbohydrate recognition domain. Gal-1 has been implicated in certain biological processes including regulation of apoptosis, cell transformation, cell adhesion, cell migration and thereby affecting the process of tumor metastasis. However, whether Gal-1 involved in oral tumorigenesis and in myofibroblasts trans-differentiation is still unknown. In this study, by using Gal-1 knockout (Gal-1-KO) mice, we analyzed whether loss of Gal-1 might decrease the incidence of carcinogen 4-Nitroquinoline-1-oxide (4-NQO) induced oral cancers. We found that the incidence of leukoplakia and oral malignancy in wild type was higher than Gal-1-KO mice. On the other hand, we have isolated healthy oral gingival fibroblasts (HGFs) and oral cancer-associated fibroblasts and demonstrated the expression of Gal-1 was higher in CAFs than in HGFs. Down-regulation of Gal-1 expression in CAFs using a lentivirus RNA interference could reduce α-smooth muscle actin, a marker of myofibroblasts. Conditioned media (CM) from CAFs increased oral cancer cells migration and invasion. Silencing of Gal-1 in fibroblasts/ myofibroblasts efficiently inhibited CM-induced cancer cells migration and invasion. Mechanism studies revealed that knockdown of Gal-1 in CAFs decreased the levels of matrix metalloproteinase-2 (MMP2) and several important chemokines such as MCP-1 in their conditioned media. Pretreatment of the MCP-1 blocking antibody to neutralize the secreted MCP-1 in myofibroblasts conditioned medium notably diminished conditioned medium-induced cancer cells migration. Taken together, our results indicatethat Gal-1 involved in myofibroblasts differentiation, and Gal-1 overexpression in CAFs increased the production of MMP2 and chemokines and promoted cancer cells migration and invasion. The significance of this finding is that Gal-1 might play a harmful role in enhancing OSCC tumorigenesis, and in the future new preventive or therapeutic strategies could take this factor into consideration.

    中 文 摘 要 I 英 文 摘 要 II 誌 謝 IV 目錄 V 緒 論 1 一、腫瘤微環境（Tumor microenvironment） 1 二、腫瘤相關纖維母細胞 (Cancer associated fibroblasts) 2 三、Galectin-1蛋白（Galectin-1） 2 四、口腔癌（Oral cancer） 4 五、致癌劑 4-nitroquinoline-1-oxide (4-NQO) 5 六、細胞激素：單核球化學趨化蛋白-1（Monocyte Chemoattractant Protein-1；MCP-1） 6 研 究 動 機 7 材 料 與 方 法 8 一、細胞培養Cell culture 8 初代培養Primary culture 8 口腔癌細胞培養 8 二、細胞蛋白質表現分析 Protein expression assay 11 三、類病毒質體的製備與選擇 Lentivirus plasmid production and selection 14 四、類病毒載體的製備 Lentivirus vector production 15 五、類病毒載體的定量 Lentivirus vector production 16 六、類病毒載體感染技術 Lentivirus vector infection 17 七、狀態培養液的收集 Collection of conditioned medium 18 八、mRNA表現量分析 mRNA expression assay 19 九、Zymography assay 24 十、細胞移行分析 Cell migration assay 25 十一、細胞侵襲力分析 Cell invasion assay 27 十二、免疫組織化學染色 Immunohistochemistry 28 十三、細胞激素蛋白質晶片分析 Proteome ProfilerTM Human Cytokine Array Panel A Array Kit 30 十四、細胞螢光染色 Immunofluorescence 31 十五、口腔癌誘發的小鼠模式 Oral tumorgenesis in mouse model 32 十六、腫瘤生長分析 Oral tumor growth assay 33 十七、老鼠腫瘤細胞模式的建立 33 實 驗 結 果 35 一、 人類纖維母細胞的初代培養 35 二、肌纖維母細胞指標蛋白α-Smooth muscle actin（α-SMA）會高量的表現在CAF 35 三、 減少Galectin-1的表現能夠調控肌纖維母細胞transdifferentiation而造纖維母細胞形態上的改變 35 四、 減少Galectin-1的表現會使纖維母細胞的α-SMA的表現量減少 36 五、 CAF conditioned medium(CM)能夠有效促進HSC-3 移行的能力，而將CAF的Galectin-1 knockdown則其CM-induced HSC-3的移行能力亦減弱 36 六、 Galectin-1 knockdown不影響conditioned medium中分泌型galectin-1的量 37 七、Cytokines array證明HGF與CAF condition medium內cytokines的表現有差異 37 八、口腔癌細胞株（HSC-3）表面細胞激素接受器的表現 38 九、 MCP-1的中和抗體能夠有效的中和conditioned medium內的MCP-1的表現，進而抑制HSC-3的移行能力 38 十、HGF及CAF conditioned medium也能夠有效誘導HSC-3 侵襲的能力，而將Galectin-1 knockdown後則會使得HSC-3的侵襲能力受到抑制 39 十一、Galectin-1的減少會影響肌纖維母細胞/纖維母細胞的狀態培養液內MMP-2的活性與蛋白質表現 39 十二、 Galectin-1（-/-） knockout老鼠的基因型鑑定 40 十三、以4-Nitroquinoline 1-oxide 誘導老鼠（wild-type、Gal-1 knockout）口腔癌前病變與口腔癌 40 十四、來自老鼠口腔癌組織的口腔癌細胞的培養 41 十五、 4-NQO treatment後誘發Gal-1 knockout老鼠腫瘤的發生率低於wild-type老鼠 41 十六、Gal-1 knockout老鼠可延遲產生口腔癌前病變與口腔癌的時間 41 十七、 wild-type老鼠產生的口腔癌體積較大 42 十八、Kaplan-Meier analysis的結果證明Gal-1 knockout老鼠能夠有效改善整體的存活率 42 討 論 43 結 論 49 參 考 文 獻 50 圖 目 錄 54 圖一、人類纖維母細胞的初代培養 54 圖二、Galectin-1與Myofibroblast指標蛋白α-Smooth muscle actin（α-SMA）會高量表現在口腔癌相關纖維母細胞 55 圖三、Galectin-1能夠調控myofibroblasts transdifferentiation造成形態上的改變 56 57 圖四、減少galectin-1的表現，會使纖維母細胞α-SMA的表現量減少 57 圖五、口腔癌相關纖維母細胞的 conditioned medium能夠增加誘導口腔癌細胞HSC-3 migration的能力 58 圖六、Galectin-1 knockdown 的fibroblasts/myofibroblasts conditioned medium 減少誘導HSC-3移行的能力 59 圖七、分泌型的galectin-1在conditioned medium內的表現並無差異 60 圖八、Cytokines array分析HGF與CAF conditioned medium內cytokines的表現的差異 61 圖九、Cytokines array分析CAF shLuc與CAF shGal-1 conditioned medium內cytokines的表現 62 圖十、口腔癌細胞株（HSC-3）表面細胞激素接受器的表現 63 圖十一、MCP-1的中和抗體能夠有效的中和conditioned medium內的MCP-1的表現，進而抑制HSC-3的移行能力 64 圖十二、Galectin-1 knockdown 的fibroblasts/myofibroblasts conditioned medium減少誘導HSC-3侵襲的能力 65 圖十三、Galectin-1的減少會影響fibroblasts/myofibroblasts conditioned medium內MMP-2的活性與蛋白質表現 66 圖十四、Galectin-1（-/-） knockout老鼠的確認 67 圖十五、以4-Nitroquinoline N-oxide (4-NQO)誘發老鼠口腔癌的實驗時程設計 68 圖十六、4-NQO treatment後老鼠口腔癌前病變的觀察 69 圖十七、4-NQO treatment後老鼠（wild-type）口腔癌的觀察 70 圖十八、4-NQO treatment後老鼠（Gal-1 knockout）口腔癌的觀察 71 圖十九、來自老鼠口腔癌組織的口腔癌細胞的培養 72 圖二十、4-NQO treatment後誘發Gal-1 knockout老鼠口腔癌的發生率低於wild-type老鼠 73 圖二十一、Gal-1 knockout老鼠（相較於wild-type老鼠）能延遲癌前病變與腫瘤發生的時間 74 圖二十二、Kaplan-Meier analysis的結果證明Gal-1 knockout老鼠能夠有效改善整體的存活率 75 表 目 錄 76 表一、4-NQO誘導wild-type/Gal-1 knockout老鼠的平均腫瘤大小的比較 76 附 圖 一 77 附 圖 二 78 自 述 79

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