臺灣博碩士論文加值系統

癌症在目前的死亡因素中位居第一位，其中大腸直腸癌發生的機率以及死亡率逐年上升，在歐美國家當中更是比台灣高出3-4倍。在近年的研究中，蛋白酶抑制劑應用於抑制發炎反應、凝血功能以及誘導癌細胞凋亡中皆扮演著重要的角色。本研究中，探討相思樹(Acacia confusa)種子所分離的胰蛋白酶抑制劑(Acacia confusa trypsin inhibitor)；簡稱ACTI)，由A鏈及B鏈以兩對雙硫鍵連結所組成的蛋白，A鏈含136個胺基酸，B鏈含39個胺基酸，分子量19.4 kDa，屬於Kunitz-type的抑制劑。
首先將ACTI從相思樹種子中純化出來，以不同濃度的ACTI處理HT-29大腸直腸癌細胞，利用MTT試驗觀察細胞的存活率，在細胞處理48小時後，可發現ACTI 濃度在3μM時細胞的存活率下降至53%，在72小時後更可以下降至40.6%。利用Invasion test，ACTI在濃度2.5 μM處理細胞48小時後，就會抑制HT-29 cells侵入性的能力達到50%，利用Gelatin zymography 的方式可以明顯觀察到ACTI濃度在5 μM處理細胞48小時後，可以抑制MMP-2及MMP-9的表現量。接著在利用RT-PCR觀察mRNA的表現，發現MMP-2及MMP-9的mRNA層次也同樣的被抑制，因此發現ACTI對HT-29 cells生長以及抑制此細胞侵入性能力，可能經由抑制MMP-2、MMP-9的表現量而抑制HT-29 cells侵入的能力。
接著我們探討ACTI對MMP-2及MMP-9的抑制作用是否藉由抑制MAPK pathway所造成，因此利用western blotting的方式看ERK的表現量，結果顯示濃度在5 μM處理細胞48小時，磷酸化ERK的表現量明顯被抑制至25％，因此推論抑制MMP的表現量以及抑制細胞的侵入能力是抑制此pathway所造成。在誘導細胞凋亡方面，利用PI染色的方式，在ACTI濃度2.5 μM處理細胞72小時，可見約50%的細胞，進一步我們再利用western blotting的方法，可以發現ACTI會導致caspase-3活化及cytochrome c由粒腺體釋出至cytosol的量增加，因此導致細胞進行apoptosis。綜合以上結果，ACTI可以抑制大腸直腸癌細胞的侵入性，可能藉由抑制ERK的磷酸化而抑制MMP-2和MMP-9的表現量，使得細胞的侵入能力降低，ACTI誘導HT-29 cells 的凋亡路徑是藉由粒腺體釋放出cytochrome c，cytochrome c至細胞液會與Apaf-1結合，形成多聚體，並促使caspase-9與其結合形成凋亡小體，被活化的caspase-9能夠再去活化caspase-3，而導致細胞凋亡。

A trypsin inhibitor (ACTI) was isolated from Acacia confusa seeds by gel filtration, DEAE-cellulose 52 and trypsin-Sepharose 4B affinity chromatography. ACTI could inhibit invasion of the HT-29 human colon cancer cells by decreasing expression of the matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in culture medium. The phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) that are the members of the mitogen-activated protein kinase (MAPK family) involved in the up-regulation of MMPs in cells. First, using gelatin zymography observed MMP-2 and MMP-9 in culture medium expression. Second, using invasion test analyze effect of ACTI in HT-29 cells. Finally, we used Western blotting to investigate the phosphorylation of ERK1/2 expression pattern by ACTI in HT-29 cells. These results showed that an ACTI treatment may decrease the expression of MMP-2 and MMP-9 through suppression on ERK1/2 phosphorylation, which in turn led to the reduction of invasive activity in the HT-29 cells. We proposed that ACTI could decrease HT-29 cell’s invasion ability via suppressing expression of MMP-2 and MMP-9, and phosphorylation of ERK1/2.

Another in this study, The ACTI could reduce the cell viability of HT-29 cells in a time- and concentration-dependent manner by using MTT test. The morphological evidence of cells was demonstrated that processed apoptosis by using PI staining after treatment ACTI 0.5μM for 72 h. We also found that amount of cytosolic cytochrome c and active caspase-3 increased at 72 hr after ACTI treatment by Western blot analysis. Thus, the results indicated that ACTI could induce apoptosis of HT-29 cells through inhibiting phosphorylation of ERK, releasing cytochrome c from mitochondria to cytosol and activated of caspase-3.

第一章 緒 論
1.1 植物蛋白酶抑制劑(protease inhibitor)
1.2 相思樹種子胰蛋白酶抑制劑 (Acacia confusa trypsin inhibitor)
1.3 大腸直腸癌之致病因
1.4 大腸直腸癌之治療
1.5 癌細胞的侵入性與轉移
1.6 論文研究方向
第二章 研究材料與方法
2.1 ACTI之萃取[35]
2.2 蛋白質濃度測定
2.3 胰蛋白酶抑制劑活性測定法
2.4 SDS-聚丙烯醯胺板膠電泳法 (SDS-polyacrylamide slab gel
electrophoresis)
2.5 細胞培養
2.6 MTT assay (偵測細胞存活率)
2.7 西方轉漬法(Western blot analysis)
2.8 細胞液中Cytochrome c之分離
2.9 Invasion test (細胞侵入能力之試驗)
2.10 Soft agar growth assay (軟性瓊膠群落生長試驗)
2.11 Gelatin zymography (活性染試驗)
2.12 Reverse transcription-PCR (RT-PCR)
2.13 Apoptosis cells的觀察
第三章 研究結果
3.1 相思樹胰蛋白脢抑制劑(Acacia confusa trypsin inhibitor；ACTI)之純化
3.2 ACTI對HT-29 cells生長之影響---細胞毒性試驗
3.3 ACTI對HT-29 cells生長之影響---軟性瓊膠群落生長試驗 (Soft agar growth
assay)
3.4 ACTI對於HT-29 cells侵入能力之影響
3.5 探討ACTI抑制HT-29 cells之MMP-2和MMP-9的蛋白質表現量
3.6 探討ACTI抑制HT-29 cells之MMP-2和MMP-9的mRNA表現量
3.7 觀察ACTI處理HT-29 cells之磷酸化ERK1/2的影響
3.8 ACTI誘導HT-29 cells細胞進行apoptosis
3.9 Cytochrome c由粒腺體釋出至cytosol的量
3.10 ACTI對HT-29 cells Casepase-3的表現量之影響
第四章 討論與結論
圖表與圖表說明
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