臺灣博碩士論文加值系統

神經膠質瘤在原發性腦瘤中最為常見，其中有三分之二屬惡性。美國每年約有17,000 名被診斷出患有原發性腦瘤，而神經膠質瘤就佔 60 %。根據世界衛生組織分類為高惡性度屬於第三級退化性星狀細胞 (anaplastic astrocytoma, AA) 及第四級多型性神經膠質母細胞瘤 (glioblastoma multiforme, GBM) 最為普遍。腦瘤治療通常以手術切除為主，輔以化學及放射線治療。但腦瘤治療的預後不佳，特別是化學及放射線治療會產生游離自由基，不僅會殺死癌細胞，連正常細胞也會殺死。最近有研究指出，抗氧化劑能提高治療效果、降低副作用並保護正常細胞。天然植物資源豐富種類繁多，據科學證實其具保健並防止癌症在復發之潛力，且多酚具有預防癌症之效果。
本研究共採集 60 種天然植物資源製備成冷凍乾燥甲醇取物，利用人類神經膠質腦瘤細胞屬於第三級的 U-87 MG 與第四期的 GBM 8401 以 MTT 還原能力法來評估萃取物對於神經膠質瘤細胞抑制生長之作用進行篩選。其中以 YY-02 甲醇萃取物抑制率最高，接著再以不同極性之溶劑正己烷、乙酸乙酯、丙酮及甲醇依序進行分離萃取，再評估其總多酚及類黃酮之含量、抗氧化及抗脂質過氧化能力，結果以甲醇再分離萃取的效果最好，並且對此兩種人類惡化神經膠質瘤細胞均具有抑制效果並具有濃度依存性，另由細胞形態及細胞週期分析結果亦發現 YY-02 甲醇分離萃取物之抗腫瘤機制乃經由細胞凋亡之模式造成神經膠質瘤細胞死亡，因此 YY-02 將來可能成為一具有開發潛力的藥物以做為治療神經膠質瘤之新應用。

Gliomas are the most common type of primary brain tumor. Nearly two-thirds of gliomas are highly malignant lesions that account for a disproportionate shape of brain tumor-related morbidity and mortality. Approximately 17,000 primary brain tumors are diagnosed every year, and of those, about 60% are gliomas. Anaplastic astrocytoma (AA, grade III) and glioblastoma multiforme (GBM, grade IV), the high grade gliomas, are the most common type hence we employed the human malignant glioma cell lines of U87 MG (III) and GBM 8401 (IV) as target to examined antiproliferation activity by MTT reduction.
Brain tumor therapy usually gets surgical resection for the first priority and followed by radiotherapy and chemotherapy. All brain tumor therapies now usually result in the poor prognosis. Especially, radiotherapy and chemotherapy would generate free radicals, kill not only cancer cells but also healthy cells. Recent studies have demonstrated that antioxidant could enhance the therapeutic effects, decrease the side effects and protect normal tissue damage. Phytochemicals are plant-derived compounds under scientific research for potential health promoting properties and recurrence of cancer. Epidemiological and preclinical evidence suggests that polyphenolic phytochemicals possess cancer chemopreventive properties.
In this study, a high through screening method was established to investigate 60 kinds of natural botany resource on anti-brain tumor activity. YY-02 methanol extract (ME) is the most efficacy of antiproliferation on human malignant glimoas. Furthermore, ME was fractionated with n-hexane (HeF), ethyl acetate (EA), acetone (AcF) and methanol (MeF). Outcomes of total polyphenol, flavonoid content, and DPPH radical scavenged, chelated ferrous ion, lipid peroxidation (LPO) inhibition of liver, kidney and brain tissue measurement, that exerted the MeF is a highest potential fraction in YY-02. Otherwise, view of morphological assay and cell cycle analysis, YY-02 exhibited the cell death pathway by apoptosis. In conclusion, YY-02 is a phytochemical of chemotherapy agents on brain tumor.

前言-------------------------------------------------------1
材料及儀器-------------------------------------------------9
一、試藥與試劑---------------------------------------------9
二、實驗用儀器--------------------------------------------14
三、樣品萃取與分離----------------------------------------15
四、細胞株------------------------------------------------17
五、試液之配製--------------------------------------------18
實驗方法--------------------------------------------------23
一、細胞株之繼代培養--------------------------------------23
二、細胞凍存----------------------------------------------23
三、冷凍細胞之活化----------------------------------------23
四、腦細胞初代培養----------------------------------------24
五、對正常腦細胞及人類神經膠質瘤細胞之增生試驗------------24
六、細胞之鏡檢反應分析------------------------------------26
七、細胞週期分析------------------------------------------26
八、總多酚量之測定----------------------------------------27
九、總黃酮類量之測定--------------------------------------27
十、抗氧化活性評估----------------------------------------28
1.清除 DPPH 自由基能力之測定------------------------------28
2.還原能力之測定(ferric reducing antioxidant power, FRAP)-29
3.螯合亞鐵離子 (ferrous ion, Fe2+ ) 能力之測定------------30
4.清除超氧陰離子 (superoxide radical, O2• -) 能力之測定--31
5.抑制黃嘌呤氧化酶 (XOD) 活性之檢測-----------------------32
6.脂質過氧化 (lipid peroxidation, LPO) 活性---------------33
十一、統計分析--------------------------------------------38
結果------------------------------------------------------39
一、60種台灣原生種植物對人類腦瘤及正常腦細胞之毒性結果----39
二、YY-02 誘導人類腦瘤細胞凋亡----------------------------45
三、YY-02 總多酚及黃酮類之含量----------------------------54
四、YY-02 自由基清除及抗氧化能力之測定結果----------------57
1.清除 DPPH 自由基之能力----------------------------------57
2.螯合亞鐵離子之能力--------------------------------------59
3.還原能力 (FRAP)-----------------------------------------61
4.清除超氧陰離子之能力------------------------------------62
5.抑制 XOD 之能力-----------------------------------------63
6.對於腦均質液，YY-02 能抑制 Fe(NH4)2(SO4)2．6H2O 誘導
所產生之脂質過氧化，但本身卻不誘導脂質過氧化------------64
7.抑制 FeCl2 誘導肝/腎粒線體脂質過氧化之能力--------------67
綜合以上結果----------------------------------------------72
討論------------------------------------------------------73
參考文獻--------------------------------------------------78
附錄------------------------------------------------------89

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