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系統識別號 U0026-2408201018471100
論文名稱(中文) EIPA經Clathrin相關之內吞作用降解NHE2而卸除懸浮癌細胞表面Fibronectin基質之組裝
論文名稱(英文) EIPA Disassembles Fibronectin Matrices of Suspended Metastatic Cancer Cells via Clathrin-related Endocytosis and Degradation of NHE2
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 98
學期 2
出版年 99
研究生(中文) 王怡方
研究生(英文) Yi-Fang Wang
學號 s1697108
學位類別 碩士
語文別 中文
論文頁數 76頁
口試委員 指導教授-鄭宏祺
口試委員-張明熙
口試委員-張敏政
口試委員-莊偉哲
口試委員-凌斌
中文關鍵字 纖連蛋白基質  鈉氫離子交換蛋白  網格蛋白  內吞作用  懸浮癌細胞 
英文關鍵字 EIPA  Fibronectin  NHE2  Clathrin  Suspended Metastatic Cancer Cells 
學科別分類
中文摘要 抑制癌症轉移發生可望延長癌症患者的存活。已知癌症細胞表面纖連蛋白基質(Fibronectin Matrix;FN matrix)促使癌症轉移。減少FN matrix組裝可望降低轉移,所以了解癌症細胞表面FN matrix組裝機制有助發展抗癌症轉移策略。過去我們發現癌症細胞膜上的穿膜蛋白-第二型鈉氫離子交換蛋白(sodium/hydrogen exchanger isoform 2;NHE2)可能當作細胞膜上FN的受器(receptor)來協助FN matrix組裝,也發現到NHE抑制劑(5-ethylisopropyl amiloride; EIPA)會抑制FN matrix組裝。在此,我們希望提供對NHE2與FN交互作用更多的證據以及了解EIPA以何種機制抑制FN matrix。首先我們針對NHE2與FN交互作用方面,從免疫螢光染色我們發現NHE2與FN matrix有共位情形。構築NHE2 extracellular domain融合蛋白(NHE2 EXs),再利用pull down assay和competition assay發現NHE2 EX2~EX5含有部分FN結合位點,這些結果提供更進一步的證據說明NHE2是FN的receptor。接著我們探討EIPA抑制FN matrix組裝機制。文獻指出EIPA抑制NHE2活性會改變細胞內pH值。為了探究是否因pH值改變而影響FN matrix組裝,我們刻意改變細胞內pH值並無影響FN matrix的組裝,推測EIPA可能藉由影響NHE2其他機制抑制FN matrix組裝。我們發現EIPA處理後的細胞NHE2明顯減少,RT-PCR確認EIPA並沒有減少細胞NHE2 mRNA,故NHE2僅在蛋白層級受到EIPA的影響。配合siRNA減少NHE2 降低FN matrix組裝的結果,推測EIPA經由降低細胞表面NHE2而阻止FN透過與NHE2結合在細胞表面組裝FN matrix。文獻指出細胞可經細胞吞噬降解來減少FN matrix及NHE在細胞膜上的表達。我們利用帶有標定的FN發現EIPA會促進FN matrix吞噬,也發現參與細胞吞噬作用的網格蛋白(Clathrin)在細胞膜周邊聚集成囊泡,推測EIPA可能促使NHE2及其結合的FN一起被吞噬進而減少細胞表面FN matrix。為探究上述假說,我們分別處理PAO(吞噬抑制劑)與NH4Cl (降解抑制劑)阻止吞噬降解作用,發現若受EIPA處理的細胞有先經過抑制劑PAO或NH4Cl之前處理其受EIPA影響的現象有中和(neutralize)的情形。綜合上述結果可知soluble form的FN的確會藉由與細胞表面NHE2的結合而促使FN matrix的組裝,並且EIPA減少FN matrix是因為EIPA促使細胞經與clathrin相關的吞噬作用,降解NHE2蛋白減少FN結合到膜上進行組裝同時也吞噬了FN matrix。
英文摘要 Tumor metastasis often causes the death of cancer patients. Understanding the molecular mechanisms underlying tumor metastasis will provide potential drug targets. In our previous studies, we found that fibronectin (FN) matrices assembed on suspended cancer cell surfaces promoted cancer metastasis. We also identified sodium/hydrogen exchanger 2 (NHE2) as a putative transmembrane receptor binding to which soluble FN is able to be incorporated into the polymeric FN matrices. Here, we further provide evidence that NHE2 is directly involved in regulating FN matrix assembly by showing that NHE2 colocalized with pericellular FN matrices and the 2nd through 5th extracellular domains of NHE2 were collectively responsible for the binding of soluble FN. Like the effect of NHE2 RNAi, FN matrix assembly was significantly impeded with NHE specific inhibitor (5-Ethylisopropyl Amiloride; EIPA). Interestingly, we noticed that the effect of EIPA was not due to its lowing the intracellular pH, but to its decreasing the protein levels of NHE2 expression. Given the fact that mRNA levels of NHE2 were not affected by EIPA treatment and that the FN matrices tend to be renewed by redering the old matrices dynamically endocytosed, we examined the possibility that EIPA exerts its effect by promoting endocytosis and degradation of surface-expressed NHE2, thus leading to FN matrix endocytosis. Indeed, we found that EIPA promoted endocytosis of FN matrices as well as NHE2. More importantly, the clustering of clathrin, often involved in clathrin-dependent endocytosis of cell surface receptors, at the subplasma membrane region was enhanced upon NHE2 treatment. These EIPA effects were able to be neutralized with endocytosis inhibitors, e.g.NH4Cl and PAO. Altogether, these data suggest the EIPA effects on reducing pericellular FN matrix are resulted from clathrin-related endocytosis and degradation of NHE2, to which soluble FN binds.
論文目次 中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖表目錄 XI
附圖表目錄 XII
縮寫表 XIII
第一章 緒論 1
1-1癌症轉移 1
1-2 臟器特異性 2
1-3 纖連蛋白 (Fibronectin; FN) 2
1-4 鈉氫離子交換蛋白 (Sodium/Hydrogen Exchanger; NHE) 3
1-5 細胞吞噬作用 (Endocytosis) 5
1-6 網格蛋白 (Clathrin) 5
1-7 研究動機 6
第二章 材料與方法 7
2-1 實驗材料 7
2-1-1 勝任細胞 (competent cell) 7
2-1-2 實驗細胞株 7
2-1-3 實驗動物 7
2-1-4 小鼠siRNA 7
2-1-5 抗體 7
2-1-6 酵素 9
2-1-7 蛋白質 9
2-1-8 培養液 10
2-1-9 緩衝液 11
2-2 實驗方法 16
2-2-1 萃取RNA 16
2-2-2 逆轉錄聚合酵素連鎖反應(RT-PCR) 17
2-2-3 PCR反應 18
2-2-4 萃取細胞蛋白質 19
2-2-5 SDS-PAGE 蛋白質電泳法 20
2-2-6 西方墨點法 (Western Blotting) 21
2-2-7 懸浮細胞免疫螢光染色 22
2-2-8 電穿孔法細胞轉染 23
2-2-9 構築MBP重組蛋白 24
2-2-10 表現及純化MBP 重組蛋白 26
2-2-11 生物素標記fibronectin 28
2-2-12 親和性沉澱試驗 28
2-2-13 EIPA與吞噬抑制劑處理細胞 29
2-2-14 Brdu細胞增生實驗 30
2-2-15 細胞黏著實驗 30
2-2-16動物模式癌症轉移實驗 31
2-2-17 Competition assay 32
第三章 結果 34
3-1 在MTF7與LLC中NHE2是主要表現的 NHE isoform 34
3-2 NHE抑制劑減少FN matrix組裝進而阻斷癌症轉移 35
3-3 NHE2與FN matrix在細胞膜上有共位現象 35
3-4 NHE2與FN有直接的交互作用 36
3-5 EIPA減少FN matrix並非因細胞內pH值改變所影響 37
3-6 EIPA減少NHE2在蛋白層級 37
3-7 EIPA促使細胞吞噬FN matrix 38
3-8 EIPA增多clathrin聚集成囊泡 39
3-9 細胞吞噬抑制劑可中和EIPA對MTF7的影響 40
第四章 結論 41
第五章 討論 42
5-1 Clathrin對NHE2、FN matrix內吞作用是否必需 42
5-2 直接辨認NHE2 extracellular domains方法 42
5-3 NHE2上與FN作用確切的結合位點 43
5-4 建立穩定knock down NHE2細胞株 44
5-5 EIPA可能以其他機制抑制FN matrix 45
5-6 細胞內吞作用與抗癌的應用 45
5-7 EIPA目前在臨床的應用 46
第六章 參考文獻 47
第七章 實驗圖表 52
第八章 附圖表 69
第九章 自述 76

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