臺灣博碩士論文加值系統

背景/目的：肺癌已成為全世界最常見的死亡原因之一。雖然肺癌常在臨床上使用化學療法，但它對身體仍有極其嚴重的毒性和副作用。褐藻糖膠是從棕色海藻中提取的多醣，具有廣泛的藥理學性質。本研究的目的是研究從墨角藻提取的褐藻糖膠協同低濃度阿黴素抑制抗藥性人肺腺癌細胞（A549/Doxo）的增殖和機制，我們希望減少化療藥物對人體的毒性和副作用，達到天然提取物輔助化療的功效。
方法：通過化療藥物阿黴素對非小細胞肺癌細胞株進行長期刺激，建立對阿黴素具有抗藥性的細胞株後，將天然提取物褐藻糖膠與抗藥性肺癌細胞株共同處理。透過MTT assay確認褐藻糖膠是否增強阿黴素對於抗藥性肺癌細胞的感受性。使用Wound healing assay觀察褐藻糖膠抑制抗藥性肺癌細胞株之遷移能力，並透過gelatin zymography assay來檢測褐藻糖膠是否影響抗藥性肺癌細胞MMP的表現量， DNA fragmentation、apoptosis assay確認褐藻糖膠是否誘導抗藥性肺癌細胞走向細胞凋亡路徑，而抗藥性肺癌細胞的抗藥性基因sorcin與細胞自噬相關基因LC3和Beclin-1以及增值相關基因β-catenin的表達透過RT-PCR去進行檢測，利用Western blot analysis確認褐藻糖膠對於抗藥性肺癌細胞增值相關的β-catenin蛋白之表現量。
結果：MTT assay的結果顯示，褐藻糖膠確實可以協同增加阿黴素對於抗藥性肺癌細胞的感受性，而Wound healing assay同樣觀察到褐藻糖膠協同阿黴素抑制抗藥性肺癌細胞的遷移能力，並且透過gelatin zymography assay觀察到褐藻糖膠能夠協同阿黴素抑制MMP-9的表現量，且通過DNA fragmentation assay和apoptosis assay觀察到褐藻糖膠協同阿黴素誘導增加凋亡，也透過RT-PCR證實褐藻糖膠協同阿黴素抑制LC3和Beclin-1、β-catenin之mRNA的表現量，Western blot analysis證實褐藻糖膠透過抑制β-catenin達到抗藥性肺癌細胞的死亡。
結論:褐藻糖膠能夠增強阿黴素去抑制抗藥性肺癌細胞，藉由降低MMP-9之表現量來抑制細胞遷移，透過誘導增加細胞凋亡的發生，以及抑制抗藥性肺癌細胞之自噬作用，並且透過降低β-catenin來抑制抗藥性肺癌細胞的增值。

Background/Aims: Lung cancer is one of the leading causes of global deaths. Though lung cancer often used chemotherapy in clinical, it still has extremely serious toxicity and side effects on the body. Fucoidan is a polysaccharide extracted from Fucus vesiculosus that has a wide range of pharmacological properties. The purpose of our present study was to investigate fucoidan enhance low concentration doxorubicin to inhibit chemodrug doxorubicin resistance of human lung adenocarcinoma cell (A549) proliferation and mechanism, we hope to reduce the toxicity and side effects of chemotherapeutic drugs on the human body and achieve the efficacy of natural extracts in adjuvant chemotherapy.
Methods: Long-term stimulation of non-small cell lung cancer cell lines by chemotherapy drugs doxorubicin, to establish a cell line resistant to doxorubicin, and then the natural extract fucoidan is co-treated with drug-resistant cell lines. It was confirmed by MTT assay whether fucoidan enhanced the sensitivity of doxorubicin to drug-resistant lung cancer cells. Wound healing assay was used to observe the migration ability of fucoidan to inhibit drug-resistant lung cancer cell lines, and the expression of MMP in drug-resistant lung cancer cells treated with fucoidan was detected by gelatin zymography assay. DNA fragmentation assay and apoptosis assay were confirmed whether fucoidan to induce the apoptosis pathway of drug-resistant lung cancer cells, the expression of the resistance-related gene sorcin and the autophagy-related genes LC3 and Beclin-1 and the proliferation-related gene β-catenin in drug-resistant lung cancer cells were detected by RT-PCR. Western blot analysis was confirmed whether fucoidan induced drug-resistant lung cancer cells to express the proliferation-related protein β-catenin.
Result: MTT assay was shown that fucoidan indeed can synergize with doxorubicin increase sensitivity of drug-resistant lung cancer cells. Wound healing assay also was observed that fucoidan synergizes increased the ability of doxorubicin to inhibit the migration of drug-resistant lung cancer cells, and through DNA fragmentation assay and apoptosis assay was observed that fucoidan synergizes with doxorubicin induce increased apoptosis. Gelatin zymography assay was demonstrated that fucoidan synergizes doxorubicin inhibited expression of MMP-9. It was also demonstrated by RT-PCR that fucoidan synergizes with doxorubicin inhibited the expression of LC3 and Beclin-1. Western blot analysis was confirmed that fucoidan synergizes doxorubicin reached the death of drug-resistant lung cancer cells by inhibiting β-catenin.
Conclusion: Fucoidan can enhance doxorubicin to inhibit drug-resistant lung cancer cells, inhibiting cell migration by decreasing the expression of MMP-9, increasing apoptosis of induction, and inhibiting autophagy in drug-resistant lung cancer cells, and inhibiting the proliferation of drug- resistant lung cancer cells by decreasing β-catenin.

摘要 ii
ABSTRACT iv
圖目錄 x
表目錄 xi
第一章 文獻探討 1
第一節 肺癌細胞 1
第二節 阿黴素 1
第二節 腫瘤抗藥性 2
第三節 可溶性抗性相關鈣結合蛋白(sorcin) 2
第四節 天然藥物 3
第五節 褐藻糖膠 4
第六節 癌症的轉移 5
第七節 細胞凋亡 6
第八節 細胞自噬 6
第九節 Wnt/β-catenin 信號傳遞路徑 8
第二章 研究動機與目標 9
第三章 研究材料與方法 11
第一節 實驗材料 11
一、細胞株 11
二、細胞培養基 11
三、實驗藥品 12
四、實驗儀器 12
五、實驗抗體 12
六、實驗試劑 13
第二節 實驗方法 14
一、褐藻糖膠的製備 14
二、細胞培養 14
三、細胞冷凍 14
四、細胞解凍 15
五、MTT assay 15
六、Wound healing assay 16
七、Gelatin zymography assay 17
八、DNA fragmentation assay 18
九、Apoptosis assay 19
十、Reverse transcription polymerase chain reaction 20
十一、Western blot analysis 21
十二、統計分析 23
第四章 研究結果 24
第一節Doxorubicin對於抗藥性肺癌細胞A549與肺癌細胞A549的影響 24
一、抗藥性肺癌細胞A549與肺癌細胞A549之細胞型態差異。 24
二、Doxorubicin對於抗藥性肺癌細胞A549和肺癌細胞A549的存活影響 25
三、Doxorubicin對於抗藥性肺癌細胞A549的遷移能力影響 25
第二節 抗藥性肺癌細胞A549和肺癌細胞A549的sorcin表現量差異 25
第三節 不同濃度之Fucoidan對於肺癌細胞A549存活率的影響 26
第四節 不同濃度之fucoidan對於抗藥性肺癌細胞A549/Doxo存活率的影響 26
第五節 不同濃度之fucoidan合併doxorubicin對於抗藥性肺癌細胞A549/Doxo存活率的影響 27
第六節 不同濃度之fucoidan對於人類胎兒肺纖維細胞株MRC-5存活率的影響 27
第七節 不同濃度之fucoidan以及doxorubicin合併fucoidan對於抗藥性肺癌細胞A549/Doxo遷移能力的影響 28
一、不同濃度之fucoidan對於抗藥性肺癌細胞A549/Doxo遷移能力的影響 28
二、Doxorubicin合併不同濃度之fucoidan對於抗藥性肺癌細胞A549/Doxo遷移能力的影響 28
第八節 不同濃度之fucoidan對於抗藥性肺癌細胞A549/Doxo MMP表現量的影響 29
第九節 Doxorubicin對於肺癌細胞A549以及不同濃度之fucoidan合併doxorubicin對於抗藥性肺癌細胞A549/Doxo細胞凋亡的影響 29
第十節 Fucoidan合併doxorubicin對於抗藥性肺癌細胞A549/Doxo細胞凋亡的影響 30
第十一節、不同濃度之fucoidan合併doxorubicin對於抗藥性肺癌細胞A549/Doxo細胞自噬基因的影響 30
第十二節、不同濃度之fucoidan合併doxorubicin對於抗藥性肺癌細胞A549/Doxo細胞增值基因的影響 31
第十三節、不同濃度之fucoidan合併doxorubicin對於抗藥性肺癌細胞A549/Doxo細胞增值蛋白的影響 32
第五章 討論與結論 33
參考文獻 37
附圖 42
附表 63
附錄 64

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