Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51263
DC FieldValueLanguage
dc.contributor.advisorGow-Chin Yenen_US
dc.contributor.advisor顏國欽zh_TW
dc.contributor.authorKao, Tzu-Cheinen_US
dc.contributor.author高滋鍵zh_TW
dc.contributor.other中興大學zh_TW
dc.date2007zh_TW
dc.date.accessioned2014-06-06T08:54:03Z-
dc.date.available2014-06-06T08:54:03Z-
dc.identifier.urihttp://hdl.handle.net/11455/51263-
dc.description.abstract傳統中醫上使用甘草已有千年之歷史,其可以被用來治療各類疾病,如普通感冒、肝病及發炎等。甘草中主要之活性成分為甘草甜素 (glycyrrhizic acid),近來研究已證實其對各種肝腎疾病皆有保護效果。部分研究亦指出甘草甜素能降低因缺血損傷所造成之動物器官損傷,並抑制初代神經元中之 NF-κB 活化,可能具有減緩因巴金森氏症 (Parkinson''s disease) 等神經細胞相關疾病之功效。因此,本研究中以大鼠腎上腺嗜鉻瘤 PC12 細胞作為體外實驗之細胞平台,以缺血以及 6-Hydroxydopamine (6-OHDA) 之傷害以模擬巴金森氏症之損傷模式,並介入甘草甜素及甘草次酸 (18β-glycyrrhetinic acid) 探討其保護細胞之能力及可能作用機制。 在血清及葡萄糖移除誘導之缺血傷害結果顯示,甘草甜素 (3.1-12.5 μM) 介入 12 小時後可顯著提昇 PC12 細胞內之 glutathione peroxidase (GPx) 及 catalase 之活性 (p<0.05),進而降低細胞內之 ROS 含量,抑制由過多 ROS 造成之細胞損傷。同時也能顯著提昇 gamma-glutamylcysteine synthetase (γ-GCS) 之 mRNA 表現量 (p<0.05),對細胞內 GSH 平衡具有助益。甘草甜素在介入3 小時後可顯著降低 Bax/Bcl-2 之比值,進而抑制粒線體膜電位 (mitochondrial membrane potential) 下降,並降低下游 caspase-9 及 caspase-3 之活化 (p<0.05),抑制由粒線體引發之細胞凋亡。 以 6-OHDA 誘導之巴金森氏症傷害結果顯示,甘草甜素及甘草次酸皆可在 6-OHDA 誘導之 PC12 細胞損傷下,顯著提昇其存活率 (p<0.05)。此外,甘草甜素及甘草次酸與 6-OHDA 共培養 6 小時後可提昇細胞中之 Bcl-2 蛋白表現,並降低 Bax/Bcl-2 之比值。甘草甜素及甘草次酸介入4 小時後亦皆可顯著提昇細胞內 PI3K 及 p-Akt 之表現量 (p<0.05)。以 PI3K 之專一性抑制劑 (LY294002) 預處理後,甘草甜素及甘草次酸對細胞之保護效果有降低之趨勢。進一步探討在神經細胞中 neutral sphingomyelinase (N-SMase) 活性。結果證實,甘草甜素及甘草次酸之介入,則能顯著降低 6-OHDA 誘導 PC12 細胞之 N-SMase 之活性 (p<0.05)。 綜合以上結果,甘草甜素及甘草次酸對 PC12 細胞對於缺血損傷以及類巴金森氏症損傷模式下,具有提昇細胞內 Bcl-2 表現量以降低Bax/bcl-2比值。以及活化細胞內 PI3K/Akt 路徑以提昇下游之 GPx 及 catalase 活性和 γ-GCS 之 mRNA 表現量,進而降低細胞內 ROS 含量並減低細胞內 N-SMase 活性,抑制細胞之凋亡。本研究顯示,甘草甜素及甘草次酸具有具有保護神經細胞因缺血及神經毒素誘導凋亡之效果。zh_TW
dc.description.abstractLicorice is a common herb plant that has been used in traditional Chinese medicine for thousand years. The active compound in licorice, glycyrrhizic acid, has been used to treat many liver diseases. Some studies also demonstrate that glycrrhizic acid can improve the recovery of kidney and heart tissues from ischemic damage and block the activation of NF-κB in primary neurons. This suggests that GA can protect against Parkinson''s disease-induced cell damage. In this study, rat pheochromcytoma PC12 cells were used as a model to evaluate neuroprotective activities of glycyrrhizic acid (GA) and 18β-glycyrrhetinic acid (18GA) against oxidative stress-induced damage in PC12 cells. Result of ischemic injury experiment induced by serum/glucose deprivation showed that cells co-incubated with GA (3.1-12.5 μM) for 12 h could significantly (p<0.05) decrease the intracellular ROS levels and increase the glutathione peroxidase (GPx) and catalase activity that leading to prevent the cell damage cause by ROS overproduction. It also increased the mRNA expression of gamma-glutamylcysteine synthetase (γ-GCS) and modulated intracellular GSH content. After co-incubated with GA for 3 h, Bax/Bcl-2 ratio was significantly (p<0.05) decreased, and led into the decrease of caspase-9 and caspase-3 activity. This phenomenon indicated that serum/glucose deprivation induced the disruption of mitochondria and resulted in apoptosis. Result of 6-Hydroxydopamine (6-OHDA) induced neural cell damage mimic neurotoxin-related Parkinson's disease showed that GA and 18GA could significantly (p<0.05) protect cells against 6-OHDA-induced cytotoxicity. GA and 18GA also decreased the Bax/Bcl-2 ratio by increasing the level of Bcl-2 after co-incubation for 6 h. Furthermore, after 4 h of co-incubation, GA and 18GA increased the expression of PI3K and p-Akt. Pre-treatment of PI3K inhibitor (LY294002) with sample and 6-OHDA proved that the protective ability of GA and 18GA certainly regulated the pathway. Moreover, the activity of neutral sphingomyelinase (N-SMase) was also tested. The result demonstrated the elevation the activity of N-SMase that stimulated by 6-OHDA was attenuated by GA and 18GA. In conclusion, GA and 18GA could decrease the Bax/Bcl-2 ratio by increase the Bcl-2 expression and protect PC12 cell from ischemic and neurotoxin induced cell injury by maintaining the integrity of mitochondria. Otherwise, GA and 18GA also increased the activities of GPx and catalase by modulating PI3K/Akt pathway, and elevated the mRNA level of γ-GCS, which led to decrease of intracellular ROS levels and N-SMase activity to prevent cell apoptosis. Result also demonstrated the attenuated N-SMase after GA and 18GA treatment. All data suggest that GA and 18GA treatment could protect the PC12 cells from apoptosis after ischemic and neurotoxin induced cell injury.en_US
dc.description.tableofcontents中文摘要............................................................................................. i 英文摘要............................................................................................. iii 目次..................................................................................................... v 表次..................................................................................................... viii 圖次..................................................................................................... ix 附圖..................................................................................................... xi 前言…………………………………………………......................... 1 第一部份 文獻整理……………………………………………… 2 壹、神經相關疾病及其相關研究………………………………… 3 ㄧ、神經缺血損傷………………………………………………. 3 二、巴金森氏症 (Parkinson’s disease)……….………………… 6 三、PC12細胞株………………………………………………… 8 貳、甘草及其相關研究……………………………………………. 10 一、甘草內成份………………………………………………… 10 二、甘草應用及研究....................................................................... 11 參、訊息傳導路徑............................................................................. 16 一、N-SMase……………………………………………………... 16 二、Phosphoinositide 3-kinase (PI3K) pathway………………… 18 三、粒線體調控之細胞凋亡路徑................................................... 21 肆、研究目的..................................................................................... 26 伍、本論文之研究架構..................................................................... 27 第二部份 甘草甜素對血清及葡萄糖移除傷害之PC12細胞之細胞保護效應................................................................ 28 摘要................................................................................................... 29 前言.................................................................................................. 30 實驗架構........................................................................................... 32 材料與方法....................................................................................... 33 結果................................................................................................... 43 一、甘草甜素對於血清及葡萄糖移除之 PC12 細胞之影響............................................................................................. 43 二、甘草甜素對於血清及葡萄糖移除之 PC12 細胞中之核染質影響..................................................................................... 43 三、甘草甜素對於血清及葡萄糖移除之 PC12 細胞內ROS之影響..................................................................................... 43 四、甘草甜素對於血清及葡萄糖移除之 PC12 細胞中抗氧化系統之影響............................................................................... 44 五、甘草甜素提昇血清及葡萄糖移除之 PC12 細胞中粒線體膜電位....................................................................................... 45 六、甘草甜素對於血清及葡萄糖移除之 PC12 細胞中caspase-9 及 caspase-3 之影響............................................. 45 七、甘草甜素對於血清及葡萄糖移除之 PC12 細胞中凋亡相關蛋白 Bax 及 Bcl-2 比值之影響....................................... 46 討論................................................................................................... 47 第三部份 甘草甜素及甘草次酸對 6-Hydroxydopamine 誘導PC12細胞毒性之可能保護機轉探討........................... 62 摘要................................................................................................... 63 前言................................................................................................... 64 實驗架構........................................................................................... 66 材料與方法....................................................................................... 67 結果................................................................................................... 73 一、6-OHDA 對 PC12 細胞之毒性濃度效應............................. 73 二、甘草甜素及甘草次酸對於 6-OHDA 傷害之 PC12 細胞存活率之影響......................................................................... 73 三、甘草甜素及甘草次酸對於 6-OHDA 傷害之 PC12 細胞內核染質之影響..................................................................... 73 四、甘草甜素及甘草次酸對於 6-OHDA 傷害 之 PC12 細胞內 Bcl-2 及 Bax 之影響..................................................... 74 五、甘草甜素及甘草次酸對於 6-OHDA 傷害之 PC12 細胞內 PI3K/Akt 路徑之探討..................................................... 74 六、甘草甜素及甘草次酸對於 6-OHDA 傷害之 PC12 細胞內 N-SMase 活性之探討..................................................... 75 討論................................................................................................... 76 總結論............................................................................................... 91 參考文獻........................................................................................... 93 表次 第一部份 表1-1、甘草之研究概況.................................................................. 15 表1-2 真核細胞中之PI3K家族.................................................. 19 表1-3、主要神經退化性疾病之功能蛋白與粒線體有關者........ 25 第二部份 表 2-1. The primer sequence and product size for RT-PCR experiment………..................................................…...... 42 圖次 第一部份 圖 1-1. PC12 細胞株...................................................................... 9 圖 1-2.甘草及其切片..................................................................... 10 圖 1-3a.甘草甜素之結構............................................................... 14 圖 1-3b.甘草次酸之結構............................................................... 14 圖 1-4. 中性神經鞘磷脂酶之活或及凋亡之調控........................ 17 圖 1-5. PI 激素系統....................................................................... 20 圖 1-6. 粒線體在自由基團代謝上之重要性................................ 22 圖 1-7. 粒線體在凋亡中之重要性................................................ 24 第二部份 圖 2-1. 甘草甜素對血清及葡萄糖移除之 PC12 細胞存活率之影響…………...………………………………………. 51 圖 2-2、血清及葡萄糖對造成 PC12 細胞核染質之濃縮以及甘草甜素對其現象之抑制……............................................ 52 圖 2-3. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內 ROS生成之影響........................................................................ 53 圖 2-4. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內 GPx活性之影響………............………………………….…... 54 圖 2-5. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內catalase 活性之影響…………………………………... 55 圖 2-6. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內 GRd活性之影響…..…..…………………………………….... 56 圖 2-7. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內 γ-GCS mRNA 表現之影響…….……………………… 57 圖 2-8. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內粒線體膜電位之影響…………………………………….…... 58 圖 2-9. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內caspase-9 活性之影響………………………………….. 59 圖 2-10. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內caspase-3 活性之影響…………………..……………… 60 圖 2-11. 甘草甜素對血清及葡萄糖移除之 PC12 細胞內Bax/Bcl-2 比例之影響………………………...……... 61 第三部份 圖 3-1. 6-OHDA 對 PC12 細胞之細胞毒性………………...… 81 圖 3-2. 甘草甜素對於 6-OHDA 誘導之 PC12 細胞存活率之影響…………………………………………………...…. 82 圖 3-3. 甘草次酸對於 6-OHDA 誘導之 PC12 細胞存活率之影響…………………………………………………….... 83 圖 3-4. 6-OHDA 對造成 PC12 細胞核染質之濃縮以及甘草甜素及甘草次酸對其現象之抑制………..…………….. 84 圖 3-5. 甘草甜素及甘草次酸對於 6-OHDA 誘導之 PC12 細胞內 Bax 及 Bcl-2 蛋白表現量之影響………………. 85 圖 3-6. 甘草甜素及甘草次酸對於 6-OHDA 誘導之 PC12 細胞內 PI3K 蛋白表現量之影響……….……...………… 86 圖 3-7. 甘草甜素及甘草次酸對於 6-OHDA 誘導之 PC12 細胞內 p-Akt 蛋白表現量之影響………………..………. 87 圖 3-8. PI3K 抑制劑對於甘草甜素保護 6-OHDA 誘導之PC12 細胞毒性之作用…………………….……..…….. 88 圖 3-9. PI3K 抑制劑對於甘草次酸保護 6-OHDA 誘導之PC12 細胞毒性之作用….……………………….……... 89 圖 3-10. 甘草甜素及甘草次酸對於 6-OHDA 誘導之 PC12 細胞內 N-SMase 活性之影響…………………..……. 90 附圖 附錄、甘草甜素及甘草次酸之可能神經保護路徑........................ 92zh_TW
dc.language.isoen_USzh_TW
dc.publisher食品暨應用生物科技學系zh_TW
dc.subject甘草甜素zh_TW
dc.subjectglycyrrhixic aciden_US
dc.subject甘草次酸zh_TW
dc.subject缺血損傷zh_TW
dc.subject巴金森氏症zh_TW
dc.subjectPC12細胞zh_TW
dc.subjectROSzh_TW
dc.subjectPI3K/Akt pathwayzh_TW
dc.subjectantioxidant enzymezh_TW
dc.subjectN-SMasezh_TW
dc.subject18β-glycyrrhetinic aciden_US
dc.subjectischemicen_US
dc.subjectPC12en_US
dc.subjectROSen_US
dc.subjectPI3K/Akt pathwayen_US
dc.subjectantioxidant enzymeen_US
dc.subjectN-SMaseen_US
dc.titleGlycyrrhizic acid and 18β-glycyrrhetinic acid protect against ischemic and 6-hydroxydopamine induced damage and its possible mechanism in rat pheochromocytoma PC12 cellsen_US
dc.title甘草甜素及甘草次酸對缺血及6-hydroxydopamine誘導PC12細胞損傷之保護作用及其可能作用機轉zh_TW
dc.typeThesis and Dissertationzh_TW
Appears in Collections:食品暨應用生物科技學系
文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.