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博碩士論文 etd-0327120-160536 詳細資訊
Title page for etd-0327120-160536
論文名稱
Title
檜木醇經由細胞外調節激酶及蛋白激酶B路徑抑制乙醯肝素酶並減少腫瘤轉移
Hinokitiol reduces tumor metastasis by inhibiting heparanase via extracellular signal–regulated kinase and Protein kinase B pathway
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
69
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2020-06-01
繳交日期
Date of Submission
2020-04-27
關鍵字
Keywords
細胞外訊號調節激酶、蛋白激酶 B、檜木醇、腫瘤轉移、乙醯肝素酶
extracellular signal- regulated kinase, protein kinase B, tumor metastasis, hinokitiol, heparanase
統計
Statistics
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The thesis/dissertation has been browsed 5693 times, has been downloaded 55 times.
中文摘要
乙醯肝素酶會藉由切斷硫酸乙醯肝素使細胞外基質瓦解,導致細胞侵襲與轉移的發生,為癌症病患高死亡率的主要原因,目前臨床使用的抗癌藥物有許多是植物來源的天然產物,例如檜木醇,過去文獻發現檜木醇在抗發炎及抗氧化過程中扮演重要角色,亦可抑制腫瘤成長及分化以促使細胞凋亡或自噬。因此本研究藉由檜木醇來探討其對小鼠黑色素瘤細胞 (B16F10) 及小鼠乳腺癌細胞 (4T1) 乙醯肝素酶表現量的影響,以及檜木醇在抑制癌症癌路徑的調控,再藉由傷口癒合試驗及 Transwell migration assay 檢測添加檜木醇後是否能使癌細胞產生抗轉移效果,最後由動物實驗觀察檜木醇對小鼠身上的腫瘤大小、分化程度、轉移情形等是否產生影響。我們的結果顯示,檜木醇能夠藉由降低蛋白激酶 B及細胞外調節蛋白激酶的磷酸化程度來抑制乙醯肝素酶的表達,進而抑制腫瘤細胞轉移。
Abstract
Heparanase cleaves the extracellular matrix by degrading heparan sulfate that ultimately leads to cell invasion and metastasis; a condition that causes high mortality among cancer patients. Many of the anticancer drugs available today are natural products of plant origin, such as hinokitiol. In the previous report, it was revealed that hinokitiol plays an essential role in anti-inflammatory and anti-oxidation processes and promotes apoptosis or autophagy resulting to the inhibition of tumor growth and differentiation. Therefore, this study explored the effects of hinokitiol on inhibiting cancer pathway in murine melanoma (B16F10) and breast (4T1) cancer cells, with emphasis on heparanase expression. We examined whether hinokitiol can exert anti-metastatic effects on cancer cells by wound healing and Transwell migration assays. In vitro cell migration assay showed that heparanase downregulation by hinokitiol led to a decrease in metastatic activity. Besides, our results showed that hinokitiol can inhibit the expression of heparanase by reducing the phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Furthermore, mouse tumor model was revealed that hinokitiol can inhibit tumor generates in vivo, which is consistent with the findings in the in virtro study.
目次 Table of Contents
Verification Letter from the Oral Examination Committee……………………...………i
Acknowledgement…………………………………………………….………........…..ii
Chinese Abstract………………………………………………………………………..iii
English Abstract…………………………………………...……………...………...…..iv
Contents……………….………………………………………………………………....v
List of Fugures……………….…………………………………………………….........ix
English Abbreviation….……………………………………………………….....……...x
I. Introduction……………………………………………………………………………1
1. Literature review………………………………………………………………...1
1.1. Tumor microenvironment (TME)…………………………...……………1
1.2. Tumor metastasis and extracellular matrix…………………….………....2
1.3. Heparanase…………………………………………………………….…5
1.3.1. Discovery of heparanase.…………………………………………...5
1.3.2. Biological functions of heparanase and therapeutic applications…..6
1.4. Hinokitiol, a potential anticancer drug……………………………...…....8
1.4.1. Medical applications of plants……………………………………...8
1.4.2. Hinokitiol……………………………………………………………9
2. Research motivation and purpose……………………………………..………..10
II. Material and method………………………………………………………………...12
1. Material………………………………………………………………………....12
1.1. Cell line……………………………………………………………..…..12
1.2. Reagent……………………………………………………………..…...12
1.3. Antibody………………………………………………………………...15
1.4. Equipment……………………………………………………………….16
1.5. Instrument……………………………………………………………….18
2. Method………………………………………………………………………….19
2.1. Cell culture……………………………………………………………...19
2.1.1. Growth Condition………………………………………………..19
2.1.2. Cell subculture…………………………………………………...19
2.1.3. Cryopreservation…………………………………………………20
2.1.4 Thawing frozen cell……………………………………………….20
2.1.5. Cell counting……………………………………………………..21
2.2. Gene transfection………………………………………………………..21
2.3. Protein extraction and quantitative analysis…………………………….22
2.4. Western blot analysis……………………………………………………23
2.4.1. Gel preparation……………………………………......…………23
2.4.2. Sample preparation and antibody………………………………..23
2.5. Wound healing assay……………………………………………………24
2.6. Transwell assay…………………………………………………………25
2.7. Animal study………………………………………………………….…25
2.7.1. Tumor treatment and survival analysis………..…………………25
2.7.2. Tumor weight and histochemical staining……………………….26
2.8. Statistical Analysis……………………………………………………...26
III. Result……………………………………………………………………………….27
1. Cell Viability and Heparanase Expression in B16F10 and 4T1 following Hinokitiol Treatment…………………………………………………………...27
2. Hinokitiol Inhibits Tumor Cell Migration…………………...…………………28
3. Hinokitiol Reduced Heparanase Expression through Protein Kinase B (AKT) and Extracellular Signal-Regulated Kinase (Erk) Signaling
Pathways…...…....29
4. Hinokitiol Elicits Suppressive Effect on Protein Expression in Tumor Cells Bearing Constitutively Active-Phosph-Protein Kinase B (P-Akt) and
Resveratrol-induced Phosph- Extracellular Signal-Regulated Kinase (P-Erk).................................30
5. Hinokitiol Reduces Cell Migration Tumor Cells Bearing Constitutively Active Phosph-Protein Kinase B (P-Akt) and Resveratrol-induced
Phosph- Extracellular Signal-Regulated Kinase (P-Erk)………………………………...32
6. Hinokitiol Inhibits Tumor Metastasis and Prolongs Survival rate in vivo……..34
IV. Discussion………………………………………………………………………….35
V. Conclusion…………………………………………………………………………..41

Reference……………………………………………………………………………….42
Figures…………………………………………………………………………….……51
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