臺灣博碩士論文加值系統

阿滋海默症（Alzheimer's disease ； AD）是一種神經退化性的疾病，阿茲海默症臨床症狀包括記憶的喪失、認知的失調，此疾病好發於老年時期。而阿茲海默症患者，不管是遺傳性的或自發性的都會發現由乙型-澱粉蛋白（β-amyloid；Aβ）沉積於病患腦中。Aβ主要是由細胞膜上的澱粉樣蛋白前驅蛋白 ( Amyloid precursor protein；APP ) 酵素切割後產生39到 43個胺基酸大小的片段。由Aβ聚集形成fibrillar Aβ會刺激中樞神經系統中的微小膠細胞（microglia cell）活化並吞噬這些fibrillar Aβ，同時高度表現inducible nitric oxide synthase ( iNOS ) 產生神經毒性物質nitric oxide ( NO ) 以及發炎性細胞激素如TNF-α 與 IL-1β等。目前在阿茲海默症的治療策略包括利用non-steroidal anti-inflammatory drugs ( NSAIDs ) 抑制 Aβ 所誘導的發炎反應及 NO 的釋放，也有研究指出，使用抗發炎的藥物可預防阿茲海默症的發生，因此研發能抑制 iNOS 基因表現之化合物，將有助於改善阿茲海默症先導藥物之開發。此研究中利用人類微小膠細胞株 ( C13NJ cells ) 為標的細胞，以 Aβ25-35 刺激C13NJ細胞，誘使其表現 iNOS mRNA 以建立篩藥模式，並利用由本校化學系周善行教授提供之合成化合物 ( No.2642 – No.2648 ) 進行藥物篩選，目前得到下列結果： (1)以20 μM Aβ25-35 活化C13NJ 細胞2小時，利用Reverse transcription-polmerase chain reaction ( RT-PCR ) 方法，可明顯偵測到 iNOS mRNA 於 C13NJ 細胞中表現；(2)在20 μM Aβ25-35 刺激12小時後，利用Western blotting方法，可偵測到 iNOS 蛋白質於 C13NJ 細胞中表現；(3)以 Aβ25-35 刺激 iNOS 基因表現為篩藥模式，目前發現編號 No.2644、 No.2647 及 No.2648 之化合物，能降低 iNOS mRNA 於C13NJ 細胞中表現；(4)藉由Alamar blue方法分析，No.2644、 No.2647及 No.2648 不具有細胞毒性；(5)於低、中、高不同濃度情況下，No.2644 與 No.2648 可劑量性降低由Aβ25-35 所誘導的 iNOS 基因於 C13NJ 細胞中之表現；(6)為了瞭解 Aβ25-35 是經由哪些訊息傳導因子活化而刺激 iNOS 基因於 C13NJ 細胞表現，以做為探討三個合成化合物作用機轉之依據，實驗中以 Aβ25-35 刺激情況下，加入Phosphoinositide 3-kinase ( PI3K )、NF-κB 與 Mitogen activated protein kinases ( MAPK ) 等抑制劑於 C13NJ 細胞中，檢測 iNOS mRNA 表現量是否受影響。結果顯示， p38、JNK 與 NF-B 抑制劑可降低 iNOS 基因之表現，得知 Aβ25-35 主要經由 p38、JNK 與 NF-B 活化途徑，而促使了iNOS 基因於 C13NJ 細胞中表現；(7) 為了確認Aβ25-35 是否能活化 C13NJ 細胞中 NF-B 訊息傳導途徑，實驗中以 Western blotting 偵測 IB 蛋白質之降解，結果發現 Aβ25-35 於刺激 C13NJ 細胞 20 分鐘後，能引起 IB 蛋白質顯著降解； (8)為了確認Aβ25-35 是否能活化 C13NJ 細胞中 MAPK 訊息傳導途徑，實驗中以 Western blotting 進行偵測，結果發現 Aβ25-35 於刺激 C13NJ 細胞 20 分鐘後，皆能引起 p38、JNK 與 ERK 之活化。(9)實驗中以 Western blotting偵測化合物 NO.2644及NO.2648 對 IB 降解的影響，結果顯示 2 化合物對於 Aβ25-35 所引起的C13NJ 細胞中IB 降解具有抑制作用。(10) Western blotting結果顯示， NO.2644及NO.2648 對於Aβ25-35 所引起 C13NJ 細胞中與p38活化皆無顯著抑制作用。由此研究成果顯示，NO.2644及NO.2648主要藉由降低 IB 降解以抑制 NF-kB 活化，繼而降低 iNOS 基因於 C13NJ 細胞中表現。藉由此研究進行，也建立了一個神經保護先導藥物篩選平台，未來可提供更多藥物之篩選。

Alzheimer's disease ( AD ) is a progressive neurodegenerative disorder and characterized with the cerebral deposition of senile plaque which arises from the abnormal accumulation of -amyloid ( A). A is a peptide of 39–43 amino acids from cleavage of the amyloid precursor protein ( APP ). Several evidences indicate that A-activated microglia cells are involved in the neuropathology observed in AD. High levels of inducible nitric oxide synthase ( iNOS ) and nitric oxide ( NO ) have been detected in microglial cells of AD patients and cause neurons death. Epidemiological studies have shown that non-steroidal anti-inflammatory drugs ( NSAIDs ) prevent or delay the onset of AD. To identify potential lead compounds with neuron protection activities, a human microglial cell line, C13NJ, was used as target cells and A-stimulated cells to express iNOS mRNA as an drug-screening model. The following results were obtained: (1) Data of reverse transcription- polymerase chain reaction (RT-PCR) demonstrated that iNOS mRNA could be induced by 20 M A at 2 hr postactivation. (2) Results of Western blotting shown that iNOS proteins expressed in A-activated C13NJ cells at 12 hr postactivation. (3) The data from RT-PCR indicated that there were 3 synthetic compounds (No. 2644, No. 2647, and No. 2648) to downregulate iNOS mRNA expression in C13NJ cells stimulated with A (4) To elucidate Ainduced iNOS mRNA expression in C13NJ cells by which signaling pathways, various inhibitors including phosphoinositide 3-kinase ( PI3K ), NF-κB, and mitogen activated protein kinases ( MAPK ) inhibitors were added into C13NJ cells then iNOS mRNA expression was determined by RT-PCR. The results indicated that p38, JNK, and NF-B inhibitors decreased iNOS gene expression in C13NJ cells stimulated by A. (5) To confirm whether A stimulated NF-B activation, the IB degradation was analyzed with Western blotting. The data shown that IB was significantly degraded at 20 min postactivation. (6) To elucidate whether A. stimulated MAPK activation, the phosphorylation of p38, JNK, and ERK was assayed by Western blotting. The results demonstrated that the phosphorylation of p38, JNK, and ERK could be significantly induced at 20 min postactivation. Based on these results, I suggest that p38, JNK, and NF-B activation are involved in iNOS gene expression in C13NJ cells induced by A. (7) The data of Western blotting indicated that No.2644 and No.2648 increased the levels of IB proteins in A -activated C13NJ cells. (8) Both No.2644 and No.2648 did not affect phosphorylation of JNK and p38 in C13NJ cells induced by A. I suggest that No.2644 and No.2648 inhibit iNOS gene expression in C13NJ cells by modulation of NF-B activation. Furthermore, I set up a neuron protection drug-screening platform in this study. This model can be used to screening more bioactive lead compounds in future.

縮簡表　-----------------------------------------------------------------2
中文摘要　--------------------------------------------------------------4
英文摘要　--------------------------------------------------------------7
壹、緒論　--------------------------------------------------------------9
貳、材料與方法　----------------------------------------------------17
參、結果　-------------------------------------------------------------25
肆、討論　-------------------------------------------------------------35
伍、參考文獻　-------------------------------------------------------41
表　----------------------------------------------------------------------49
圖　----------------------------------------------------------------------51

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