臺灣博碩士論文加值系統

人類軟骨肉瘤是一種惡性初級的骨頭腫瘤，多好發於成年與老年，化學療法和放射性治療對其治療成效不大，特點為高度惡性和轉移潛能。此外有文獻證實許多癌細胞皆會有高表現量的內皮素-1 (Endothelin-1; ET-1)
。ET-1為具有多種調節功能的細胞激素，屬於一種分泌型的細胞激素，會和ETAR和ETBR (endothelin A and B receptor)結合進而調控人類癌症細胞的增生、血管新生、轉移及侵襲等。然而ET-1對於人類軟骨肉瘤轉移的機制目前還有很多的不清楚。我們發現ET-1會促進人類軟骨肉瘤細胞移行的能力、增加上皮-間質轉化(Epithelial-Mesenchymal Transition；EMT)和EMT轉錄因子(TWIST)的表現。前處理ETAR和ETBR抑制劑(BQ123和BQ788)、AMPK抑制劑(AraA和compound C)以及AMPK siRNA α1和α2會抑制ET-1增加人類軟骨肉瘤移行能力和TWIST的表現。在ET-1的刺激下會增加AMPK磷酸化。除此之外，microRNA(miRNA) 300 mimic也會降低ET-1增加細胞移行能力和TWIST的表現。另外，ET-1會抑制miRNA-300結合到TWIST mRNA上進而調節TWIST的表現。綜合以上結果，ET-1促進TWIST表現經由與ETAR和ETBR的結合並透過AMPK的訊息傳導進而下調miRNA-300，最後造成人類軟骨瘤細胞株EMT的發生和移行能力。

Chondrosarcoma is a malignant primary bone tumor associated with adult and the aged that responds poorly to both chemotherapy and radiation therapy. It is characterized by high malignant and metastatic potential. Recently, some studies found that endothelin-1(ET-1) is highly expressed in many cancer cells. However, the effects of ET-1 on cell motility and epithelial-mesenchymal transition (EMT) and TWIST expression in human chondrosarcoma cells are largely unknown. Here we found that ET-1 induced the cell motility, EMT and expression of TWIST in human chondrosarcoma cells. Endothelin A and B receptor (BQ123 and BQ788), AMPK inhibitor (AraA and compounpC), AMPK siRNA α1 and α2 inhibited ET-1-induced cell motility and TWIST expression. ET-1 stimulation increased the phosphorylation of AMPK. In addition, microRNA(miRNA)-300-mimic also antagonized ET increased cell motility and TWIST expression. Moreover, ET-1 induced the binding of miR-300 to the TWIST mRNA to down-regulated TWIST expression. Taken together, our results suggested that ET-1 enhances motility and EMT of chondrosarcoma cells by increasing TWIST expression through the ETAR, ETBR and AMPK signal transduction pathway and down-regulating miR-300.

目錄 I
英文縮寫 V
中文摘要 VI
英文摘要 VII
壹、緒論 1
一、軟骨肉瘤： 1
(一)、何謂軟骨肉瘤： 1
(二)、軟骨肉瘤的診斷： 1
(三)、軟骨肉瘤的分級： 2
(四)、軟骨肉瘤的治療： 2
(五)、軟骨肉瘤與轉移： 3
二、轉移(Metastasis)： 4
三、上皮-間質轉化(Epithelial-Mesenchymal Transition；EMT)： 5
(一)、EMT簡介： 5
(二)、EMT的功能： 5
(三)、EMT與癌症： 6
四、AMP活化蛋白??(AMP-activated protein kinase；AMPK)： 7
(一)、AMPK的作用： 7
(二)、AMPK對於癌症： 7
五、TWIST相關蛋白1(TWIST-related protein1；TWIST1)： 8
(一)、何謂TWIST： 8
(二)、TWIST與癌症： 8
六、內皮素-1(Endothelin-1；ET-1)： 9
(一)、ET-1簡介： 9
(二)、ET-1的作用： 9
(三)、ET-1與癌症： 10
七、MicroRNAs(miRNAs)： 12
(一)、何謂MicroRNA： 12
(二)、MiRNA以及癌症： 12
貳、研究架構與動機 26
一、研究背景： 26
二、研究目的： 26
?礡B材料與方法 27
一、實驗材料： 27
(一)、試劑： 27
(二)、儀器設備： 28
二、實驗方法： 29
(一)、細胞培養(Cell Culture)： 29
(二)、細胞傷口癒合分析(Wound-healing assay)： 29
(三)、細胞移行分析(Migration assay)： 30
(四)、細胞侵襲分析(Invasion assay)： 30
(五)、即時聚合?○s鎖反應(Quantitative Real-Time PCR)： 30
(六)、細胞轉染(Transfection)： 32
(七)、西方點墨法(Western Blot)： 32
(八)、免疫組織染色(Immunohistochemistry；IHC)： 33
(九)、冷光 (Luciferase) 活性測試： 33
(十)、統計方法： 34
肆、結果 35
一、ET-1增加人類軟骨肉瘤移行能力和EMT表現量： 35
二、ET-1透過內皮素受體(ETAR或者ETBR)導致人類軟骨肉瘤細胞移行的能力和EMT表現量： 35
三、ET-1透過轉錄因子TWIST調節調節人類軟骨肉瘤細胞移行的能力和EMT表現： 36
四、AMPK參與人類軟骨肉瘤細胞的移行能力和EMT表現： 36
五、ET-1透過下調miR-300來調節人類軟骨肉瘤細胞的移行能力和EMT表現： 37
六、在人類軟骨肉瘤病患中癌症期別、ET-1和TWIST表現的相關性： 37
七、高度轉移以及一般的軟骨肉瘤細胞之間移行能力和EMT表現的差異： 38
伍、討論 39
陸、結論 41
柒、參考文獻 42


圖目錄
Figure 1 Enchondromas, arising from deregulated growth plate differentiation caused by constitutively active hedgehog signaling, can progress to low-grade central chondrosarcoma when additional genetic changes occur 13
Figure 2 Chondrosarcoma is the second most frequent primary malignant tumor of bone 14
Figure 3 The metastatic cascade 15
Figure 4 Dissemination of cells from a primary solid tumor is facilitated by the epithelial–mesenchymal transition (EMT) 16
Figure 5 Different types of EMT 17
Figure 6 Schematic overview of EMT pathways. 18
Figure 7 Components of the endothelin system 19
Figure 8 Disease processes in which endothelin-1 / endothelin ETA-receptor signaling has been demonstrated to play a role 20
Figure 9 The ET1 regulated tumor–microenvironment interactions in tumor maintenance and progression 21
Figure 10 The ET1 signaling network 22
Figure 11 Canonical miRNA biogenesis pathway 24
Figure 12 MicroRNAs (miRNAs) as tumor suppressors and oncogenes 25
Figure 13 ET-1 promotes cell migration and EMT of chondrosarcoma cells. 50
Figure 14 ET-1-directed migration activity of human chondrosarcoma. 52
Figure 15 ET-1 mediated cell migration of chondrosarcoma cells through increasing TWIST expression. 54
Figure 16 AMP-activated protein kinase (AMPK) is involved in cell migration and ET-1-induced EMT expression, examine by treating with inhibitor. 56
Figure 17 AMP-activated protein kinase (AMPK) is involved in cell migration and ET-1-induced EMT expression. , examine by treating with siRNA. 58
Figure 18 ET-1 promotes cell migration and EMT expression by down　regulating microRNA (miRNA)-300 expression. 60
Figure 19 The miR-300 directly represses the TWIST expression through binding to the 3’UTR of the human TWIST. 62
Figure 20 The correlation among ET-1, TWIST, tumor stages in human chondrosarcoma tissues. 64
Figure 21 Highly metastatic cell (JJ-S10 cell) induce metastasis, EMT, ET-1 expression and suppress miR-300 expression. 66
Figure 22 Schema of signaling pathways involved in ET-1 induced EMT expression and metastasis in chondrosarcoma cells. 67

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