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研究生:許祐輔
研究生(外文):Yu-Fu Hsu
論文名稱:經由刺蝟及類鐸受體四訊息路徑所影響的肝細胞癌及巨噬細胞的交互作用
論文名稱(外文):The interaction of hepatocellular carcinoma and macrophages through sonic hedgehog and TLR4 signaling pathway
指導教授:曾炳輝
指導教授(外文):Ping-Hui Tseng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:47
中文關鍵詞:肝細胞癌巨噬細胞類鐸受體四刺蝟訊息路徑
外文關鍵詞:Hepatocellular carcinomaMacrophageTLR4Sonic hedgehog
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一、 緒論................................................ 1
1-1 肝細胞癌................................................ 1
1-2 腫瘤微環境與巨噬細胞................................ 1
1-3 肝細胞癌與巨噬細胞的交互作用................................ 2
1-4 類鐸受體四................................................ 3
1-5 類鐸受體四訊息路徑在肝細胞癌當中的角色....................... 3
1-6 刺蝟訊息路徑................................................ 4
1-7 Sonic hedgehog訊息路徑在肝細胞癌當中的角色................ 4
1-8 CCL2於巨噬細胞與肝癌當中的角色................................ 5
二、 研究目的與動機................................................ 6
三、 實驗材料與方法................................................ 7
3-1 實驗材料................................................ 7
3-1-1. 細胞株及培養液................................................ 7
3-1-5. Reagent................................................ 8
3-2 實驗方法................................................................ 10
3-2-1. 細胞培養與繼代 Cell culture................................ 10
3-2-2. RNA萃取及即時聚合酶連鎖反應 RNA extraction and real-time PCR (q-PCR)..... 10
3-2-3. 細胞遷移實驗 Transwell migration assay................ 11
3-2-4. 細胞共培養實驗 Transwell Co-culture assay................ 11
3-2-5. 細胞存活率實驗 MTT assay................................ 11
3-2-6. 西方點墨法前蛋白質樣品製備 Protein sample preparing................ 12
3-2-7. 西方點墨法 Western blot................................ 12
3-2-8. 慢病毒製備與感染 Lentivirus production and infection................ 12
3-2-9. 酵素結合免疫吸附分析法 Enzyme-linked immunosorbent assay (ELISA)....... 13
3-2-10.細胞計數 Cell counting................................ 13
四、 實驗結果................................................ 14
4-1 以LPS刺激HCC細胞株及巨噬細胞株模擬發炎環境................ 14
4-2 測試HCC細胞株被LPS刺激活化後是否招募更多巨噬細胞................ 14
4-3 觀察利用sh-RNA病毒默化HCC細胞株及巨噬細胞株後對招募巨噬細胞的影響............ 15
4-4 測試巨噬細胞被LPS刺激活化TLR4後是否影響HCC細胞株的遷移能力................16
4-5 觀察共培養系統中HCC細胞株的細胞活性變化 .........................16
4-6 觀察共培養系統中HCC細胞株的細胞凋亡訊息路徑是否活化................ 17
4-7 觀察共培養系統中HCC細胞株與巨噬細胞的NF-κB及P38訊息路徑是否活化........ 17
4-8 觀察共培養系統中HCC細胞株與巨噬細胞的趨化因子及細胞激素的表現.......... 19
五、 結論與討論................................................ 20
5-1 結論................................................ 20
5-2 討論................................................ 21
六、 實驗圖表................................................ 24
圖1. 觀察Huh7及THP-1利用LPS刺激後的基因表現................ 25
圖2. 從Huh7所收集的conditional medium提升招募巨噬細胞的能力............. 26
圖3. Huh7及THP-1默化結果................................ 27
圖4. 默化Huh7的GLI1所收集的培養液顯著降低THP-1遷移數量................ 29
圖5. THP-1細胞及培養液提升Huh7遷移能力................................ 31
圖6. 共培養系統中Huh7細胞活性降低................................ 34
圖7. 共培養系統中Huh7的細胞凋亡路徑活化................................ 35
圖8. 共培養系統中Huh7及THP-1的炎症相關路徑................ 36
圖9. 共培養系統中Huh7及THP-1的TNF-a、IL-6、CCL2 及GLI1基因表現......... 38
圖10. 共培養系統Huh7培養液中的CCL2表現上升................................ 39
圖11. 使用低劑量LPS刺激PLC5細胞模擬慢性發炎................ 40
附錄................................................ 41
附錄圖1. 腫瘤微環境中巨噬細胞的來源及活化................ 41
附錄圖2. 腫瘤微環境中巨噬細胞對於腫瘤發展的影響................ 42
參考文獻................................................................ 43
參考文獻

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