臺灣博碩士論文加值系統

菸鹼醯胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide；NAD+) 在生物體內很多反應中扮演很重要的角色，例如：在氧化還原反應中扮演輔因子(cofactor)、或是在ADP核糖基化 (ADP-ribosylation) 反應中當作是受質，提供ADP核糖。而菸鹼胺磷酸核糖基轉移酶 (Nicotinamide phosphoribosyltransferase；Nampt)是菸鹼胺(nicotinamide) 經回收利用路徑生成NAD+ 的速率決定酵素，它可以調節哺乳動物中Sirt1蛋白的活性。在先前研究指出，很多癌細胞具有較快速NAD+ 代謝的情形，而FK866 是Nampt 的競爭型抑制劑，使用FK866 可以降低癌細胞中NAD+ 的含量，進而引起細胞凋亡。另外，吲哚胺2,3-二氧化酶(Indoleamine 2,3-dioxygenase；IDO) 是色氨酸(Tryptophan) 經新合成路徑合成NAD+ 的速率決定酵素，先前研究也指出在一些腫瘤模式中，IDO的抑制劑(L-1MT) 具有不錯的治療效果。因此，我們推論如果能結合Nampt inhibitor 及IDO inhibitor 或許能有更好的治療效果，根據我們推論此更好的治療效果可由以下兩個方式來達成：(1) 使用Nampt inhibitor 和IDO inhibitor 完全地阻斷腫瘤中NAD+ 的生合成，進而破壞腫瘤的生長與代謝；另外(2) 結合此兩種抑制劑或許也可以透過”避免腫瘤細胞進行免疫逃脫，而提升宿主免疫能力”來達到更好的治療效果。而在我們活體(in vivo) 試驗中發現：結合此兩種抑制劑在免疫缺乏的NOD/scid 老鼠看不到顯著的療效，但在C3H/HeNCrj 老鼠產生了很顯著延遲其腫瘤生長及提高存活率的效果。因此，我們分析腫瘤內免疫細胞如： CD4、CD8、自然殺手細胞(natural killer cell；NK) 及嗜中性白血球(neutrophil) 浸潤(infiltration)的情形，結果顯示嗜中性白血球浸潤的情形似乎跟FK866結合L-1MT 所產生的加成性治療效果較有高度相關。而先前文獻指出Nampt會促使血管新生(angiogenesis)，所以最後我們利用組織免疫染色(Immunohistochemistry, IHC) 分析老鼠腫瘤中血管新生的狀況，結果顯示FK866結合L-1MT可以有效地降低腫瘤血管生成至對照組的 45％。所以FK866結合L-1MT 可以有更好的治療效果其主要是因為FK866 可以降低腫瘤細胞中NAD+ 的含量和L-1MT 可以避免腫瘤進行免疫逃脫所達成。

Nicotinamide adenine dinucleotide (NAD+) has crucial roles in many cellular processes, such as a coenzyme for redox reactions or as a substrate to donate ADP-ribose units. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in the salvage pathway of NAD+ synthesis via nicotinamide and regulates the activity of the NAD-dependent deacetylase Sirt1 in mammalian cells. In previous study, it showed that tumor cells had a high rate of NAD+ turnover owing to elevated ADP-ribosylation activity. In addition, FK866 is a potent small-molecule inhibitor of human Nampt, and the consequent reduction in NAD+ levels can cause apoptosis of tumor cells. On the other hand, Indoleamine 2,3-Dioxygenase (IDO) is the rate-limiting enzyme in the de novo pathway of NAD+ synthesis via tryptophan. IDO inhibitors can potentiate chemotherapy in some cancer models. Hence, We hypothesized that it may enhance cancer therapy by combination of FK866 and L-1MT. We proposed that the significant effect on cancer therapy could be accomplished by one of the following: (1) blocking the NAD+ biosynthesis pathway with FK866 and L-1MT can further inhibit the growth and metabolism of tumor cells. (2) FK866 in combination with L-1MT can achieve better cancer therapy by avoiding tumor cells going to immune escape. In this study, FK866 in combination with L-1MT could significantly delay the tumor growth and elevate the survival of C3H/HeNCrj mice. In contrast, FK866 and L-1MT did not have additive effect on NOD/scid mice. We analyzed the infiltration of immune cell, including CD4, CD8, NK and neutrophil. The result showed a high relationship between neutrophil infiltration and the anti-cancer effect of FK866 in combination with L-1MT. In addition, we analyzed the new blood vessel in the mice tumor by immunohistochemistry. The result suggested that FK866 in combination with L-1MT could significantly reduce tumor angiogenesis up to 45％ when comparing with primary mouse bladder tumor cell line. In conclusion, the therapeutic efficiency may be due to in part from immune enhancement by IDO inhibitor and in part from growth inhibition by Nampt inhibitor.

緒論
ㄧ. NAD+ 和cancer的關連 1
二. Nampt 在NAD+ biosynthesis 的角色 2
三. IDO在NAD+ biosynthesis的角色 4
四. 研究目標與策略 6
材料與方法
ㄧ.細胞培養與藥物處理 8
二.質體製備 13
三. 構築HA6L-hPBEF、HA6L-hPBEF(1~1230)、HA6L-hPBEF(1~969)、HA6L-hPBEF(214~1476)、HA6L-hPBEF(448~1476)、pGBKT7-hPBEF(1~600)、pGBKT7-hPBEF(601~1476) 17
四.透析conditioned medium 24
五.西方墨點法 25
六.細胞生長分析計算 33
七.酵母雙雜交(yeast two hybrid) 33
八.動物實驗 34
九.治療效果評估方式 35
實驗結果
ㄧ.分析給予FK866及L-1MT後是否能有效地降低細胞中total NAD
(NADt) 含量 39
二. In vitro分析FK866及L-1MT對癌細胞生長的影響 39
三. FK866結合L-1MT之抗腫瘤能力評估於免疫缺乏的NOD/scid老鼠 40

四. FK866結合L-1MT之抗腫瘤能力評估於C3H/HeNCrj同源鼠
(syngeneic mice) 41
五.分析給予FK866及L-1MT之老鼠腫瘤組織免疫細胞浸潤的情形 42
六.分析給予FK866及L-1MT之老鼠腫瘤血管新生(angiogenesis)的情
形 43
七.探討哪個片段對Nampt的分泌來說是必須的 44
八.利用酵母雙雜交系統(yeast two hybrid system) 尋找Nampt 的
相互作用蛋白(interaction protein) 44
討論 46
結論 50
參考文獻 51
圖表 58
附圖 63
附註 78
自述 81

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