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研究生:陳怡伶
研究生(外文):Yi-Ling Chen
論文名稱:腫瘤細胞表現Fasligand促進其皮下腫瘤之生長,但抑制腫瘤細胞轉移至肺臟
論文名稱(外文):Fas ligand expression on tumor enhances tumorigenesis, but suppresses experimental lung metastasis in vivo
指導教授:楊倍昌楊倍昌引用關係
指導教授(外文):Bei-Chang Yang
學位類別:博士
校院名稱:國立成功大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:137
中文關鍵詞:細胞凋亡腫瘤嗜中性球皮下肺臟
外文關鍵詞:apoptosistumorneutrophillungscutaneously
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Fas和Fas ligand (FasL; CD95L; Apo-1L)的結合會誘發部份表現Fas的細胞凋亡(apoptosis)。以前的研究認為腫瘤細胞表現FasL (tumor FasL)以躲避免疫系統的攻擊。但是,腫瘤細胞表現FasL如何影響腫瘤的生長及轉移,至目前仍不清楚。本實驗利用FasL特定的核醣酵素(FasL-specific ribozyme; FasLRibozyme)來抑制FasL基因的表現,並且建立帶有FasL特定的核醣酵素的B16F10黑色素癌細胞株(FasL表現低腫瘤細胞;FasLlow tumor cells;FasLR),應用於皮下腫瘤形成及腫瘤細胞轉移至肺部的動物實驗,分析腫瘤細胞的FasL表現量和腫瘤細胞的生長和轉移之相關性。所建立的FasL表現量高(FasLhigh)和FasL表現量低(FasLlow)細胞株的Fas和腫瘤壞死因子-□(tumor necrosis factor-□; TNF-□□的基因表現量並無差異。
當腫瘤細胞株以皮下方式植入小鼠體內,雖然FasL可以促進嗜中性球浸潤及腫瘤細胞的凋亡(apoptosis)。FasL表現量低(FasLlow)細胞株在皮下形成腫瘤的速率比FasL表現量高(FasLhigh)細胞株慢。在缺乏嗜中性球小鼠的體內,FasL表現量低(FasLlow)細胞株的生長顯著大於在正常小鼠體內的生長速率。相對地,FasL表現量高(FasLhigh)細胞株腫瘤的生長,在嗜中性球正常或是缺乏的小鼠體內,並無顯著差異。局部注射脂多醣(LPS)至C57BL/6及NOD/SCID小鼠的腫瘤區域,顯著抑制FasL表現量低(FasLlow)細胞株的生長,而對於FasL表現量高(FasLhigh)細胞株的生長抑制作用較為輕微。
在生體外將腫瘤細胞和嗜中性球一起培養,嗜中性球都能毒殺FasL表現量高(FasLhigh)和FasL表現量低(FasLlow)細胞株。FasL表現量高(FasLhigh)細胞株對於嗜中性球所誘發的細胞凋亡(apoptosis),比FasL表現量低(FasLlow)細胞株具有耐受性。腫瘤細胞表現FasL會抑制嗜中性球的明膠酵素B (gelatinase B)的分泌、氧活性物質(reactive oxygen species; ROS)的產生及CD11b的表現。因此,腫瘤細胞表現FasL導致嗜中性球不活化的作用機制,可能是皮下腫瘤細胞在躲避免疫系統攻擊的原因之一。
如果將腫瘤細胞株以尾靜脈注射至小鼠體內,FasL表現量低(FasLlow)細胞株轉移至肺臟的數目明顯高於FasL表現量高(FasLhigh)細胞株。在腫瘤細胞侵入肺部時,腫瘤細胞表現FasL也能促進嗜中性球浸潤及腫瘤細胞細胞的凋亡(apoptosis)。利用抗體去除小鼠體內的嗜中性球,可以明顯促進FasL表現量高(FasLhigh)和FasL表現量低(FasLlow)細胞株轉移至肺臟。
總之,腫瘤細胞表現Fas-L的確會影響腫瘤的生長,但是其效益會因腫瘤形成的器官或組織而有不同。
Fas (CD95) and Fas ligand interaction induces apoptosis in Fas+ cells of many cell types. The expression of FasL on tumor cells (tumor FasL) has been implicated in evasion of immune surveillance. How Fas-L on tumors affects tumorigenesis and metastasis, however, is not clear. In this study, we have established B16F10 melanoma-derived cells carrying either plasmid encoding FasL specific ribozyme (FasLR; FasLlow tumor cells) or vector plasmid (FasLhigh tumor cells) to investigate the role of Fas-L in tumorigenicity and metastasis. FasLR suppressed the expression of FasL but not Fas or TNF-□ in B16F10 melanoma cells. When injected subcutaneously into C57BL/6 mice, FasLlow tumor cells grew slowly than did FasLhigh melanoma. FasLhigh tumor cells showed more intensive neutrophilic infiltration accompanied by multiple necrotizing areas than did FasLlow tumor. The average size of FasLlow tumors, but not that of FasLhigh tumors, was significantly enhanced in mice depleted of neutrophils. Consistently, a local injection of LPS to recruit/activate neutrophils significantly delayed tumor formation of FasLlow tumor cells, and slightly retarded that of FasLhigh tumor cells in both C57BL/6 and NOD/SCID mice.
Neutrophils killed FasLlow melanoma cells more effective than FasLhigh melanoma cells in vitro. The resistance of FasLhigh melanoma cells to being killed by neutrophil was correlated with impaired neutrophil activation as demonstrated by reductions in gelantinase B secretion, ROS production, and the surface expression of CD11b and the transcription of FasL. Local transfer of casein-enriched or PMA-treated neutrophils delayed tumor formation by melanoma cells. Thus, inactivation of neutrophils by tumor FasL is an important mechanism by which tumor cells escape immune attack.
On the other hand, suppression of FasL in B16F10 melanoma cells by FasLR enhanced lung metastasis of the cells in C57BL/6 mice, and that was correlated with reductions in both apoptotic tumor cells and granulocytic infiltration. Mice depleted of granulocytes, but not CD4+ and CD8+ cells, showed a greatly elevated susceptibility to lung metastasis. Moreover, apoptosis in tumor cells was significantly reduced in granulocyte-depleted mice during the course of tumor formation.
In summary, FasL on tumors affects tumorigenesis and metastasis, but the effect was diverse in different tissues or organs.
目錄………………………………………………………………………………..………..I
中文摘要…………………………………………………………………………………...V
英文摘要…………………………………………………………………………….…...VII
表目錄……………………………………………………………………………….……IX
圖目錄…………………………………………………………………………………..….X
縮寫索引…………………………………………………………………………………XII
第一章緒論……………………………………………………………………..….…….1
第一節Fas/Fas ligand 的作用………………………………………...…..….…….1
第二節腫瘤反擊假說(Tumor counterattack hypothesis)與嗜中性球(neutrophils)浸潤(infiltration) …………………...…..….………………….…………....2
第三節腫瘤細胞轉移和Fas ligand之相關性……………………….…...….…….4
第四節腫瘤細胞轉移和嗜中性球(neutrophil)之相關性………………………….7
第五節腫瘤細胞表現Fas ligand和嗜中性球活化之相關性……………………..9
第二章實驗目的與策略……..………………………………………………...……….11
第一節 重要問題……………………………..…………………..…..……….…...11
第二節FasL特定的核醣酵素(FasL-specific ribozyme; FasLRibozyme)的架構…...13
第三節小鼠黑色素癌細胞B16F10動物模式……………………………………14
第四節CD4、CD8 T細胞及嗜中性球缺失抗體之應用……………………….…15
第五節嗜中性球的活化…………………………………………………………..16
第三章實驗材料及方法……………………………………………………… …….…18
第一節材料……………………………………………………………………......18
第二節實驗流程…………………………………………………………………..22
第三節試劑溶液製備方法……………………………………………………..…38
第四章結果……………………………………………………………….………..…...45
第一節小鼠黑色素癌細胞B16F10穩定細胞株的建立……………………...…45
1.FasL特定的核醣酵素基因殖入(transfection)和FasL表現量高(FasLhigh)及FasL表現量低(FasLlow)腫瘤細胞株的篩選( selection)………...........…..45
2.確認腫瘤細胞株FasL、Fas、腫瘤壞死因子-(TNF-w、轉型生長因子-(TGF-w、及巨噬細胞發炎蛋白質-2(MIP-2)等相關基因表現量….45
3.在生體外(In vitro)腫瘤細胞的生長與死亡……………………………….46
第二節 皮下黑色素癌細胞的生長之動物模式…………………………………...47
1. 皮下腫瘤細胞的生長及小鼠存活率之觀察………………………………..47
2. 腫瘤細胞FasL的染色及病理切片之觀察…………………………………48
3. 皮下腫瘤生長的動物模式,不同時間點的嗜中性球浸潤………………..49
4. CD4、CD8 T細胞或是嗜中性球缺失之小鼠………………………………49
5. 嗜中性球的浸潤和腫瘤細胞的細胞凋亡之相關性………………………..51
6. 皮下注射脂多醣(LPS)對FasL表現量不同之腫瘤細胞的影響……….…..51
7. 局部腫瘤部位注射(adoptive transfer)嗜中性球或是PMA活化的嗜中性球…………………………………………………………………………….52
第三節 黑色素癌細胞B16F10轉移之動物模式………………………………….52
1. 腫瘤細胞轉移至肺臟及小鼠存活率之觀察………………………………..52
2. 腫瘤細胞的FasL的染色及病理切片之觀察………………………………53
3. 腫瘤細胞轉移至肺臟,不同時間點的顆粒性球(granulocytes)浸潤………54
4. CD4、CD8 T細胞或是顆粒性球缺失之小鼠………………………………55
5. 顆粒性球的浸潤和腫瘤細胞的細胞凋亡之相關性………………………..55
6. 靜脈注射(adoptive transfer)嗜中性球,對FasL表現量不同之腫瘤細胞的影響…………………………………………………………………………….56
7. 腫瘤細胞在免疫缺失NOD/SCID小鼠體內之轉移……………………….56
8. 腫瘤細胞在肺臟巨噬細胞缺失小鼠體內之轉移…………………………..57
9. 中和小鼠體內介白素-10 (IL-10)之動物實驗………………………………57
第四節 在生體外(In vitro)腫瘤細胞和嗜中性球交互作用………………………57
1. 細胞毒殺實驗(cytotoxicity)…………………………………………………57
2. 嗜中性球活化指標及氧活性物質(reactive oxygen species; ROS)的產生..58
3. 鼠科(Murine) Fas/Fc蛋白質的作用………………………………………..59
4. 嗜中性球和腫瘤細胞共同培養其Fas、轉型生長因子-(TGF-w、腫瘤壞死因子-(TNF-w、巨噬細胞發炎蛋白質-2 (MIP-2)及介白素-10 (IL-10)等相關基因表現量………………………………………………………………...59
第五章討論…………………………………………………………………....……..…61
第一節FasL表現量高(FasLhigh)和FasL表現量低(FasLlow)穩定細胞株的建立.63
1.FasL特定的核醣酵素(FasL-specific ribozyme; FasLRibozyme)抑制黑色素癌細胞FasL基因及蛋白質的表現量………………………………………..63
2.抑制黑色素癌細胞的FasL基因的表現,降低細胞內轉型生長因子-(TGF-w和巨噬細胞發炎蛋白質-2 (MIP-2)基因的表現……………64
第二節 腫瘤細胞表面的FasL造成嗜中性球功能異常,促進小鼠皮下腫瘤的生長……………………………………………………………………………66
1.腫瘤細胞表現Fas ligand,促進嗜中性球的浸潤……………..………....66
2.活化的嗜中性球抑制皮下腫瘤之生長……………………………..……..68
第三節 腫瘤細胞表面的FasL,促進嗜中性球的浸潤,抑制腫瘤細胞的轉移...69
1.表現FasL的腫瘤細胞,吸引顆粒性球浸潤至肺部,影響腫瘤細胞轉移至肺部的數量………………………………………………….…………..…69
2.顆粒性球依賴性細胞凋亡(Granulocyte-dependent apoptosis)參與腫瘤細胞的轉移………………………………………………………………………71
3.介白素-10 (IL-10)和腫瘤細胞的轉移之相關性的探討…………………..72
第四節 腫瘤細胞表現FasL之作用及可能機制之探討………………………...73
第六章 參考文獻……………………………………………………….….……..……....78
第七章 附圖及表…………………….…………………………………..…..……………98
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