臺灣博碩士論文加值系統

有許多報導指出，靈芝具有抑制腫瘤生長的能力，但大多為多醣體和三帖類。由松杉靈芝 (Ganoderma tsugae)當中發現的真菌類免疫調節蛋白稱為松杉靈芝免疫調節蛋白 (Fungal immunomodulatory protein, FIP-gts)，在此研究中我們利用已選殖此蛋白基因的重組蛋白工程在大腸桿菌中表現並純化得到reFIP-gts。但是重組FIP-gts蛋白對於癌症細胞的作用並沒有很清楚的被研究和探討。造成癌症病人惡化死亡最主要的因素之一就是腫瘤細胞具有轉移的能力，在之前使用受傷癒合分析 (wound healing assay)，已發現隨著加入重組FIP-gts蛋白濃度的增高，相對的也增強了抑制細胞移動性，本研究更進一步利用Boyden Chamber去分析證實重組FIP-gts蛋白可以抑制細胞移動 (migration)及侵襲能力 (invasion)，分別處理0.075 mM時只可以抑制11%左右的細胞移動，在抑制侵襲能力方面，分別處理0.075 mM時就可以抑制95%左右的細胞侵襲。在此時，我們也發現到重組FIP-gts蛋白有誘導Nm23 抑癌蛋白及其mRNA活化的能力，進而導致抑制細胞的轉移能力，且在過度表現Nm23蛋白的A549細胞中，也觀察到細胞的移動能力很明顯下降許多，而在gelatin-zymography中觀察到MMP-2的活性完全被抑制住。而用gelatin-zymography方式偵測MMP (Matrix metalloproteinases)活性，重組FIP-gts蛋白會抑制MMP-2的分泌量。且利用RT-PCR的方式觀察MMP的抑制劑TIMP-1和TIMP-2，發現到TIMP-1則受到重組FIP-gts蛋白增加其表現，但TIMP-2則沒有變化，而MMP-2的量同時也看到表現量的減少，因此得知重組FIP-gts蛋白能抑制MMP-2的活性，使得細胞移動性也被抑制。為了更進一步確認重組FIP-gts蛋白可以抑制細胞的轉移，我們使用FIP-gts單株抗體去中和，結果可以恢復55%左右的細胞移動，用gelatin-zymography方式發現可以恢復些許的MMP-2活性，故重組FIP-gts蛋白的確具有抑制肺癌細胞株轉移的能力。我們也利用二維電泳也觀察到A549細胞中有不同蛋白受到重組FIP-gts 蛋白刺激後而改變，在未來將再進一步確定這些蛋白為何。所以本研究闡示松杉靈芝免疫調節蛋白FIP-gts有潛力發展成臨床抑制細胞轉移能力的藥劑。

A large number of polysaccharide and triterpenoids of Ganoderma lucidum has been reported to inhibit the growth of tumors. Fungal immunomodulatory proteins, FIP-gts, were found in Ganoderma tsugae. In this study, we expressed and purified the fungal immunomodulatory protein (FIP-gts) in E. coli. However, the effect of FIP-gts on cancer cells has not been characterized clearly. Metastasis is one of the most difficult problems in cancer therapy. In wound healing assay, the level of motility have been decreased by the increasing concentration of FIP-gts in the previous study. In order to confirm the effect of FIP-gts, the Modified Boyden chamber assay was applied. Treatment of A549 cells with up to 0.07 mM of FIP-gts decreased cell migration by 11% for 8 h. 0.075 mM of FIP-gts decreased cell invasion significantly. In addition, Nm23 protein and mRNA was increased by reFIP-gts addition on A549 cells. We also observed the inhibitor of cell migration by overexpression of Nm23 protein in A549 cells. The secreted MMP-2 activity was notably inhibited measured by gelatin-zymography. Inhibitioty effect the FIP-gts on the MMP-2 activity of A549 cells in gelatin-zymography assay. In RT-PCR, the level of MMP-2 and MMP-9 were decreased but the MMP inhibitor of TIMP-2 had no effect and TIMP-1 increased after treated with FIP-gts. To further ascertain the antimetastatic activity of FIP-gts, we assessed the counteraction of FIP-gts with monoclonal FIP-gts antibody. Monoclonal FIP-gts antibody could reverse cell migration by 55%. In gelatin-zymography assay, MMP-2 activity was also slightly increased. We also observed differentproteins that changed between untreated or treated with FIP-gts in A549 cells using 2-DE gel analysis. We are going to identify these proteins in the future. Our results highlight the potential of FIP-gts in the treatment of clinical cancer metastasis.

壹、 中文摘要 9
貳、 英文摘要 (Abstract) 11
参、 縮寫表 13
肆、 緒論 (Introduction) 15
一、肺癌流行病學及危險因 15
二、腫瘤細胞的轉移 (Metastasis) 16
三、Matrix metalloproteinase (MMPs) 18
(1)基質金屬蛋白水解酵素之分類 19
(2)基質金屬蛋白水解酵素的表現、調控與活化 20
四、Tissue inhibitor of metalloproteinases (TIMPs)
介紹 21
五、靈芝簡介 21
六、松杉靈芝免疫調節功能蛋白 24
七、松杉靈芝免疫調節功能蛋白具有的生理活性 25
八、p53的介紹 26
九、Nm23/nucleoside diphosphate kinase 的發現 27
(1)Nm23蛋白的發現 27
(2)Nm23-H1於肺癌之表現 28
伍、 研究動機 (Motivation) 30
陸、 實驗材料 (Materials ) 32
一、藥品試劑 (Drugs and reagent) 32
二、實驗設備 (Instruments) 33
三、人類肺癌細胞株 (Cell lines)之來源 35
柒、 實驗方法 (Methods) 36
一、細胞培養 (Cell culture) 36
(1)細胞培養 (Cell culture) 36
(2)繼代培養 (Replate cell) 36
(3)冷凍細胞 (Frozen cell) 36
(4)解凍細胞 (Defrozen cell) 37
(5)DNA轉殖 (Transfection) 37
二、細胞傷口癒合試驗 (Wound healing assay) 39
三、細胞轉移試驗 (Modified Boyden chamber) 39
(1)Migration測試 39
(2)Invasion測試 40
四、膠菜瓊脂電泳分析 (Agarose gel electrophoresis) 41
(1)1.5 % agarose gel的製備方法 41
(2)電泳操作 41
(3)由Agarose gel回收DNA片段 (Elution) 41
五、轉形作用 (Transformation) 42
(1)勝任細胞 (Competent cells)製備 42
(2)轉形作用的步驟 43
(3)質體DNA的微量抽取 43
六、西方點墨法 (Western Blotting) 44
(1)樣品製備 (Sample preparing) 44
(2)蛋白濃度定量分析 (Protein quantification) 45
(3)SDS-聚丙烯醯胺板膠電泳之轉漬 (SDS-PAGE) 45
(4)抗體作用及偵測方法 (Antibody detection) 47
(5)磷酸化抗體作用及偵測方法
(phosphate antibody detection) 48
七、酵素活性分析 (Gelatin Zymogragphy) 48
八、酵素免疫分析法細胞激素之Human TNF-a之測定(ELISA) 50
九、反轉錄酶-聚合酶連鎖反應 (RT-PCR ) 52
(1)RNA純化 52
cDNA的合成 ( RT-PCR ) 52
(2)PCR ( 鏈聚合酶鏈鎖反應 )： 53
十、細胞貼附試驗 (Anchorage independent assay) 53
十一、二維電泳法 (2-D Electrophoresis) 54
十二、RNAi (RNA intereference) 55
捌、結果 (Results) 57
一、松杉靈芝免疫調節功能蛋白對A549肺癌細胞其移行性之
影響 57
二、松杉靈芝免疫調節功能蛋白對細胞移行性 (Metastasis)
之影響 57
三、觀察松杉靈芝對A549肺癌細胞之MMP-2的影響 58
四、觀察松杉靈芝對免疫調節功能活性之分析—釋出細胞激素58
五、觀察松杉靈芝對A549肺癌細胞中的蛋白表現和MAPK 路徑
的影響 59
(1)觀察A549肺癌細胞在處理重組FIP-gts後是否會活化p53、
p21和Nm23蛋白 59
(2)在A549肺癌細胞中FIP-gts活化MAP kinase的情形 60
六、觀察松杉靈芝對A549肺癌細胞之Nm23 mRNA 表現情形 60
七、製作穩定且過度表現Nm23蛋白的A549肺癌細胞 61
八、觀察松杉靈芝對A549和A549/Nm23肺癌細胞之細胞移行性
(Metastasis)相關的mRNA表現情形 61
九、觀察過度表現Nm23蛋白的A549肺癌細胞之MMP-2的表現
情形 61
十、松杉靈芝對A549/Nm23肺癌細胞之細胞移行性(Metastasis)
之影響 62
十一、松杉靈芝對A549肺癌細胞之細胞轉型(Transformed)之
影響 62
十二、觀察A549 和A549/Nm23肺癌細胞經由松杉靈芝抗體中
和松杉靈芝後之細胞移動和MMP-2活性的影響 63
十三、A549肺癌細胞經由松杉靈芝抗體中和松杉靈芝後之相關
蛋白表現 64
十四、觀察松杉靈芝對A549肺癌細胞蛋白表現的差異 64
十五、建構pcDNA/HU6/Nm23-H1-RNAi 64
玖、討論 ( Discussion ) 66
拾、圖表說明 (Figures and Tables) 73
Figure 1處理FIP-gts對A549細胞移動的影響
(Wound healing assay) 73
Figure 2處理FIP-gts對A549細胞移動的影響
(Modified Boyden chamber) 74
Figure 3處理FIP-gts對A549細胞侵襲的影響
(Modified Boyden chamber) 75
Figure 4處理FIP-gts對A549細胞分泌的MMP-2影響 76
Figure 5處理FIP-gts對A549細胞分泌的TNF-a影響 77
Figure 6處理FIP-gts對A549細胞分泌的IFN-γ影響 78
Figure 7處理FIP-gts誘導A549細胞中p53、p21和Nm23的情形 79
Figure 8處理FIP-gts誘導A549細胞中MAPK pathway的情形 80
Figure 9處理FIP-gts對A549細胞中Nm23 mRNA的影響 81
Figure 10轉染Nm23-flag至A549細胞的確認 82
Figure 11處理FIP-gts對A549細胞中TIMP-1、MMP-9、
TIMP-2和MMP-2 mRNA的影響 83
Figure 12A549細胞和pcDNA/A549以及Nm23/A549細胞
MMP-2蛋白的表現差異 84
Figure 13A549、pcDNA/A549和A549/Nm23細胞分泌
MMP-2的變化 85
Figure 14處理FIP-gts對A549和A549/Nm23細胞移動的影響 86
Figure 15處理FIP-gts對A549、pcDNA/A549以及Nm23/A549
細胞轉型的影響 87
Figure 16處理FIP-gts抗體對A549細胞移動的影響 88
Figure 17處理FIP-gts抗體對A549細胞分泌的MMP-2影響 89
Figure 18處理FIP-gts抗體對A549/Nm23細胞分泌的MMP-2影響90
Figure 19處理FIP-gts抗體對A549細胞之p53、p21和Nm23的
情形 91
Figure 20處理FIP-gts對A549細胞所有蛋白表現量的影響 92
Figure 21外加FIP-gts來確定FIP-gts在二維電泳中的位置 93
Figure 22建構pcDNA/HU6/Nm23-H1-RNAi 94
拾壹. 參考文獻( Reference ) 95
拾貳. 附表以及附圖 108

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