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研究生:鄭文泰
研究生(外文):Wen-tai Cheng
論文名稱:抗氧化中草藥抽取物神經保護功效之研究
論文名稱(外文):Studies on neuroprotective effect of antioxidant Chinese herb extract
指導教授:李孟真李孟真引用關係
指導教授(外文):Meng-jen Lee
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:90
中文關鍵詞:PC12細胞穿心蓮星狀膠質細胞神經保護發炎反應
外文關鍵詞:Andrographis paniculataPC12astrocyteinflammationneuroprotection
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  • 被引用被引用:3
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穿心蓮 (Andrographis paniculata Nees.) 為爵床科植物,以往被用為抗菌、消炎解熱及免疫促進之傳統治療藥物。由文獻中發現,從穿心蓮植物中可萃取出23種有效成分的純物質,這些純物質主要可分為黃酮類 (flavonoids) 和內酯類 (lactones)。其中的l4-去氧-11, 12-二去氫穿心蓮內酯 (穿心蓮丁素) (14-deoxy-11, 12-didehydroandrographolide, AP3) 為內酯類中labdane型之雙萜類活性成分,近年來陸續有研究發現穿心蓮丁素亦表現出抗凝血、降低血壓的效果。由文獻中指出,牛樟芝子實體萃取出同樣的物質 (穿心蓮丁素, AP3) 可以保護皮層神經細胞 (cortical neurons) 免予被beta amyloid所誘發的細胞死亡。因此,我們欲進一步證實並探討穿心蓮萃取物 (穿心蓮丁素, AP3) 在PC12細胞經由過氧化氫的氧化傷害模式上,是否具有抗氧化的神經保護功效。不論PC12細胞經由過氧化氫與AP3先、後或是同時處理後,經由MTT與LDH測試結果,AP3可有效阻礙過氧化氫所造成的細胞死亡,顯示AP3具有抗氧化保護PC12細胞的效果。AP3亦可有效抑制初代星狀膠質細胞經由TNF-α刺激活化釋放更多TNF-α,顯示AP3有抗發炎的效果。但是,AP3在星狀膠質細胞條件培養基 (Astrocyte condition medium, ACM) 與過氧化氫中一同處理PC12細胞,AP3卻無抗氧化神經保護PC12細胞的效果。
14-deoxy-11,12-didehydroandrographolide (AP3) is a diterpene lactone isolated from A. paniculata. In recent years several researchers have explored its different interesting biological properties such as antihepatic, antifertility, hepatoprotective, antithrombotic, immunostimulant, antihepatotoxic, antiplatelet aggregation, cardiovascular, antihyperglycaemic and antioxidant, anti-inflammatory and antimalarial. Diterpene lactones from the Fruiting Body of Antrodia camphorate are previously report protect neuron from beta amyloid induced cell death. In the present study, we examined the effect of AP3 on hydrogen peroxide (H2O2) -induced pheochromocytoma cell line PC12 cell death. The protective effect of the AP3 against H2O2-induced oxidative damage on the PC 12 was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. AP3 increased PC12 cellular viability in a dose-dependent manner. AP3 inhibit stimulated TNF-α release from primary astrocytes, but can not reduce death of PC 12 cells treated with astrocyte condition medium (ACM) and H2O2..
目錄
書名頁
博、碩士論文授權書
論文口試委員會審定書
論文口試委員會審定書英文版
中文摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VII
第一章 緒論 1
1-1 穿心蓮 1
1-1.1 穿心蓮植物學分類 1
1-1.2 穿心蓮簡介 1
1-1.3 穿心蓮植物型態特徵 1
1-1.4 穿心蓮民間傳統方例 3
1-1.5 穿心蓮萃取物有效成分 3
1-2 氧化壓力 10
1-2.1 氧化壓力形成 10
1-2.2 過氧化氫所造成之氧化傷害 11
1-2.3 抗壞血酸 12
1-2.4 氧化壓力與中樞神經系統疾病 14
1-3 發炎反應 15
1-3.1 發炎反應發生 15
1-3.2 中樞神經系統之發炎反應 15
1-3.3 α型腫瘤壞死因子 (tumor necrosis factor-α,TNF-α) 17
1-3.4 中樞神經系統內膠質細胞之發炎角色 20
1-4 細胞凋亡 21
1-4.3 細胞凋亡發生 21
1-4.4 細胞凋亡活化路徑 21
1-4.5 氧化壓力誘導細胞凋亡與神經退化性疾病 23
1-5 PC12細胞株 24
1-6 星狀膠質細胞 24
1-7 MTT分析原理 25
1-8 乳酸去氫酶分析原理 26
1-9 實驗目的 27
第二章 材料與方法 28
2-1 實驗大綱 28
2-2 實驗設備 29
2-3 實驗材料 29
2-4 實驗方法 33
2-4.1 PC12細胞 33
2-4.2 過氧化氫處理 34
2-4.3 抗壞血酸與過氧化氫處理 35
2-4.4 AP3與過氧化氫處理 35
2-4.5 初代星狀膠質細胞 36
2-4.6 星狀膠細胞條件培養基收集 37
2-4.7 AP3、過氧化氫與星狀膠質細胞培養液處理 38
2-4.8 α型腫瘤壞死因子之測定 39
2-4.9 MTT測試 40
2-4.10 乳酸去氫酶測試 (LDH assay) 40
2-4.11 BrdU assay 41
2-4.12 分析與統計方法 42
第三章 實驗結果 43
3-1 過氧化氫對PC12細胞之氧化傷害 43
3-2 抗壞血酸對PC12細胞之生理機能影響 43
3-3 穿心蓮丁素 (AP3) 對PC12細胞之生理機能影響 45
3-4 穿心蓮丁素 (AP3) 降低星狀膠質細胞經由α型腫瘤壞死因子 (TNF-α)刺激合成與釋放更多TNF-α 47
3-5 穿心蓮丁素 (AP3) 在星狀膠質細胞條件培養液 (ACM) 中對PC12細胞之生理機能影響 47
3-6 穿心蓮丁素 (AP3) 與過氧化氫同時在星狀膠質細胞條件培養液 (ACM) 中對PC12細胞之生理機能影響 48
3-7 穿心蓮丁素 (AP3) 與過氧化氫同時在TNF-α活化過的星狀膠質細胞條件培養液 (SACM) 中對PC12細胞之生理機能影響 49
第四章 討論 50
4-1 抗壞血酸 (ascorbate) 抗氧化保護與傷害PC12細胞 50
4-2 AP3抑制星狀膠質細胞經由腫瘤壞死因子-α刺激腫瘤壞死因子-α量 52
4-3 AP3具有抗氧化保護PC12細胞效果 53
4-4 AP3在過氧化氫與星狀膠質細胞培養液中無抗氧化保護PC12細胞效果 54
4-5 MTT測試實驗誤差 56
參考文獻 68
作者簡歷 90
表目錄
表1-1 穿心蓮萃取物結構 4
表1-2 穿心蓮萃取物相關活性 7
表1-3 中樞神經系統中TNF-α的產生 18
表1-4 中樞神經系統中TNF-α的作用 19
表2-1 抗壞血酸與過氧化氫處理 35
表2-2 AP3與過氧化氫處理 36
表2-3 AP3、過氧化氫與星狀膠質細胞培養液中處理 38
圖目錄
圖1-1 穿心蓮植物型態 2
圖1-2 Labdane基本骨架 4
圖1-4 中樞神經系統中免疫發炎反應 17
圖1-4 Caspase活化與細胞凋亡 22
圖1-5 MTT及MTT formazan之構造圖 26
圖1-6 LDH測量原理 26
圖3-1 過氧化氫對PC12細胞之氧化傷害 58
圖3-2 抗壞血酸對PC12細胞之生理機能影響 (MTT assay) 59
圖3-3抗壞血酸對PC12細胞之生理機能影響 (LDH assay) 60
圖3-4穿心蓮丁素 (AP3) 對PC12細胞之生理機能影響 (MTT assay) 61
圖3-5穿心蓮丁素 (AP3) 對PC12細胞之生理機能影響 (LDH assay) 62
圖3-6穿心蓮丁素 (AP3) 對PC12細胞之生理機能影響 (BrdU assay) 63
圖3-7 穿心蓮丁素 (AP3) 降低星狀膠質細胞經由α型腫瘤壞死因子 (TNF-α) 刺激合成與釋放更多TNF-α 64
圖3-8 穿心蓮丁素 (AP3) 在星狀膠質細胞條件培養液 (ACM)中對PC12細胞之生理機能影響 65
圖3-9 穿心蓮丁素 (AP3) 與過氧化氫同時在星狀膠質細胞條件培養液 (ACM) 中對PC12細胞之生理機能影響 66
圖3-10 穿心蓮丁素 (AP3) 與過氧化氫同時在TNF-α活化過的星狀膠質細胞條件培養液 (SACM) 中對PC12細胞之生理機能影響 67
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