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系統識別號 U0026-0812200911180535
論文名稱(中文) FasL對腫瘤球體的影響:細胞的移動與對免疫細胞的感受性
論文名稱(英文) FasL determines the properties of tumor spheroids:cell migration and sensitivity to immune cells
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 92
學期 2
出版年 93
研究生(中文) 王琳雅
研究生(英文) Lin-Ya Wang
學號 s4691105
學位類別 碩士
語文別 中文
論文頁數 97頁
口試委員 指導教授-楊倍昌
口試委員-許秉寧
口試委員-沈孟儒
口試委員-湯銘哲
中文關鍵字 Fas配體  腫瘤球體  腦神經膠質瘤 
英文關鍵字 FasL  tumor spheroid  glioblastoma 
學科別分類
中文摘要 在生物體中,惡性細胞所形成的腫瘤組織,是以複雜而立體的網絡結構組成,而在養分供應的機制、訊息傳遞的機制、細胞之間的接觸、以及細胞與胞外基質的交互作用等反應中,皆與正常的組織不同。在體外培養的模式中,部分腫瘤細胞在鋪有膠體的培養基上,可互相聚集,形成多細胞腫瘤球體 (multicellular tumor spheroid,MTS)。在腫瘤生物學以及器官分化學的研究方面,不同於平面培養模式,多細胞腫瘤球體培養系統提供了新的研究方向。本實驗中,利用多細胞腫瘤球體培養系統,研究Fas配體 (FasL) 對免疫監控能力及細胞移動能力的影響。Fas,又稱為CD95或APO-1,是細胞膜上的穿膜型蛋白,屬於腫瘤壞死因子接受器家族的一員。Fas配體,又稱為FasL或CD95L,是Fas的專一性配體,屬於腫瘤壞死因子家族的一員,是細胞膜上的穿膜型蛋白。Fas與FasL的交互作用,會使細胞內受質引發一系列的訊息傳遞,導致表現Fas的細胞凋亡。部分腫瘤細胞表面的FasL與免疫細胞表面的Fas結合後,可阻止免疫細胞侵入腫瘤組織,甚至引發免疫細胞的凋亡,以逃避免疫清除。人類腦神經膠質瘤細胞 (glioblastoma) 是快速生長的腦腫瘤細胞,槌頭型ribozyme對FasL具專一性,經轉染後可有效抑制腦神經膠質瘤細胞U-118MG的FasL表現。轉染Fas-L ribozyme的U-118MG細胞稱為U-118R,而轉染空載體的U-118MG細胞則作為載體控制組,稱之U-118V。轉染Fas-L ribozyme並不影響U-118R細胞形成腫瘤球體。經過混合培養後,Jurkat、BJAB、HL-60等免疫細胞皆附著於腫瘤球體表面,U-118V腫瘤球體結構被Jurkat與HL-60破壞,而U-118R腫瘤球體經混合培養後保持完整球狀結構,表示FasL的表現量影響腫瘤球體與免疫細胞之間的交互作用。利用Fas的競爭型抗體CH-11及抑制型抗體ZB-4的處理,有助於釐清FasL在腫瘤球體崩解中扮演的角色。腫瘤球體中的細胞與胞外基質結合而形成球狀架構,本實驗中發現FasL的表現量影響腫瘤球體中細胞黏附因子 (focal adhesion molecules) 的表現,並進一步調控細胞移動的能力。此外,FasL的表現量影響腫瘤球體matrix metalloproteinase (MMP) 的分泌,細胞黏附因子如何受到調控而影響細胞移動能力,以及MMP是否參與其中,需要進一步探討。本實驗結果中,推測FasL可能具有啟動免疫細胞毒殺腫瘤球體細胞的能力,另一方面則調控細胞的移動能力,有助於腫瘤細胞的移動及轉移。


英文摘要 Malignant cells in neoplastic tissue are organized in complex three-dimensional networks displaying nutrient and signal gradients, cell-cell contact, as well as cell-extracellular matrix interaction. Some tumor cell lines form multicellular tumor spheroids (MTS) in vitro when they are grown on an agar-medium base. The MTS culture system has been shown to provide new insights into tumor biology as well as organ differentiation. In this study, we used the MTS culture system to investigate the contribution of FasL for immune survaillance. Fas (CD95/APO-1) and its specific ligand, FasL (CD95L) are surface proteins of the tumor necrosis factor receptor superfamily whose interaction triggers a cascade of subcellular events resulting in apoptosis of the Fas-expressing targets including immune cells. The presence of FasL on tumor cells is thought to inactivate tumor-infiltrating lymphocytes. A hammerhead Fas-L-specific ribozyme (Fas-L ribozyme) has been used to suppress the FasL gene of U-118MG glioblastoma cells, which are rapidly growing brain tumors. U-118MG-derived cells carried Fas-L ribozyme named U-118R and carried control plasmid named U-118V. Transfection of Fas-L ribozyme did not affect the spheroid formation. Jurkat cells, BJAB cells and HL-60 cells attached well to the surface of spheroids. Only U-118V spheroids but not U-118R spheroids were destroyed by the presence of Jurkat or HL-60, demonstrating that different levels of FasL could affect the interaction between tumor cells and immune cells. To elucidate how tumor spheroids were destructed, the application of angonistic anti-Fas antibody CH-11 and antagonistic anti-Fas antibody ZB-4 demonstrated the effect between Jurkat cells and tumor spheroids. We further found that FasL modified the expression of focal adhesion molecules in spheroids, which contribute to the adhesion of cells to the extracellular matrix, and involved in the potential of cell migration. To clarify the cell migration ability of tumor spheroids, the possible involvement of focal adhesion molecules and matrix metalloproteinase (MMP) should be further investigated. Our results suggested that FasL might initiate the cytotoxic activity of immune cells against tumor spheroids, and also mediate cell migration of tumor spheroids.


論文目次 目錄

中文摘要……………………………………………………………………….1
英文摘要……………………………………………………………………….3
第一章 序論…………………………………………………………………..5
第一節 Fas與FasL的作用…………………………………………..…5
第二節 人類腦神經膠質瘤細胞 (human glioblastomas)………………6
第三節 體外立體細胞培養模式:腫瘤球體培養系統 (spheroid system)…………………………………………………………..7
第四節 FasL特定核醣酵素 (FasL-specific ribozyme,FasLribozyme) 的架構……………………………………………………………...8
第五節 FasL結合胞外基質 (extracellular matrix) 並影響腫瘤組織結構………………………………………………………………...9
第六節 Matrix metalloproteinases (MMPs) 與腫瘤生物學…………..10
第七節 FasL與免疫反應的關係………………………………………11
第二章 實驗目的……………………………………………………………13
第一節 腫瘤球體 (tumor spheroid) 體外細胞培養系統……………..13
第二節 Fas與FasL作用對腫瘤細胞的影響…………………………..13
第三節 FasL特定核醣酵素 (FasL-specific ribozyme,FasLribozyme) 的應用…………………………………………………………….14
第四節 實驗方向……………………………………………………….15
第五節 實驗流程……………………………………………………….16
第三章 實驗材料與方法……………………………………………………17
第一節 實驗材料……………………………………………………….17
第二節 實驗方法……………………………………………………….21
1. 細胞與細胞培養 (cells and cell culture)……………………….21
2. 腫瘤球體的養成與觀察 (spheroid formation and observation).23
3. 冷凍切片技術 (Frozen section technique)……………………..23
4. 蘇木紫與伊紅染色法 (Hematoxylin-Eosin staining)………….24
5. 免疫組織化學染色法 (Immunohistochemtry,IHC)…………...25
6. 抽取細胞蛋白質 (protein extraction)…………………………..26
7. 測定蛋白質濃度 (protein concentration determination)……….27
8. 西方墨點法 (Western blot analysis)……………………………27
9. 細胞移動情況分析 (migration assay)………………………….31
10. Gelatin zymography assay………………………………………31
11. 腫瘤球體與免疫細胞的混合培養 (co-culture system)………..32
12. 競爭型Fas抗體 (CH-11) 與對抗型Fas抗體 (ZB-4) 的應用.32
13. 臨界點乾燥 (Critical point drying,CPD)……………………...33
14. 掃描式電子顯微鏡技術 (Scanning Electronic Microscopy,SEM)…………………………………………………………….34
第三節 溶液配製方法………………………………………………….34
第四章 實驗結果……………………………………………………………43
第一節 U-118R及U-118V細胞及腫瘤球體型態觀察……………….43
1. U-118R及U-118V細胞型態觀察……………………………...43
2. U-118R及U-118V腫瘤球體的型態觀察與大小測定………...43
3. U-118R及U-118V腫瘤球體的冷凍切片與蘇木紫-伊紅染色..44
第二節 U-118R及U-118V細胞及腫瘤球體內細胞之移動能力…….44
1. 細胞邊界黏附分子 (focal adhesion molecule)在U-118R及U-118V腫瘤球體內的表現…………………………………….44
2. 傷口癒合分析法 (wound healing assay)……………………….45
3. 腫瘤球體黏附細胞培養盤與細胞移動之觀察………………...46
4. 分析U-118R及U-118V細胞與腫瘤球體所分泌的matrix metalloproteinase (MMP)……………………………………….46
第三節 腫瘤球體與免疫細胞混合培養之觀察與分析……………….47
1. Jurkat、HL-60、BJAB免疫細胞型態觀察………………………47
2. U-118R及U-118V腫瘤球體與免疫細胞的混合培養………...47
3. 分析混合培養液中所含的MMP表現量………………………48
4. 以Fas競爭型抗體 (CH-11) 與抑制型抗體 (ZB-4) 處理U-118R及U-118V腫瘤球體…………………………………...49
第五章 討論…………………………………………………………………51
第一節 U-118R及U-118V細胞及腫瘤球體的形成與結構………….51
1. U-118R及U-118V細胞特性的探討…………………………...51
2. U-118R及U-118V腫瘤球體的形成與結構…………………...51
第二節 U-118R及U-118V細胞與腫瘤球體所表現的focal adhesion molecules與對細胞移動的影響………………………………53
1. U-118R及U-118V細胞與腫瘤球體所表現的focal adhesion molecules………………………………………………………..53
2. FAK的表現對U-118R及U-118V腫瘤球體細胞移動的影響...54
3. MMP對腫瘤球體內胞外基質及細胞移動能力的影響……….55
第三節 U-118R及U-118V腫瘤球體與免疫細胞混和培養的探討….56
1. U-118R及U-118V腫瘤球體與免疫細胞的混和培養………...56
2. 混合培養後U-118V腫瘤球體的崩解與MMP表現的關係…..58
3. FasL與混合培養後腫瘤球體崩解的關係……………………..59
第四節 Fas/FasL的交互作用與其他細胞反應………………………..60
第六章 參考文獻……………………………………………………………62
圖表附錄……………………………………………………………………...73
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