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研究生:張念慈
研究生(外文):Nian-cih Chang
論文名稱:藍綠藻水萃物對於耳鳴抗氧化狀態及離子通道之影響
論文名稱(外文):The effects of spirulina platensis water extract on antioxidative state and ion channels in tinnitus.
指導教授:詹吟菁詹吟菁引用關係
指導教授(外文):Yin-ching Chen
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:92
中文關鍵詞:耳鳴主動迴避試驗老化促進小鼠抗氧化發炎離子通道藍綠藻
外文關鍵詞:senescence accelerated prone micetinnitusantioxidativeion channels.active avoidance testinflammatorySpirulina platensis
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水楊酸(salicylates),又稱阿斯匹靈(Aspirin),為已知會誘導耳鳴發生的藥物,而目前對水楊酸誘導耳鳴的路徑尚不清楚。耳鳴為一種聽覺上的幻覺,目前為止並無有效的治療方式。研究發現,藍綠藻具有抗氧化、抗發炎、神經保護功能。本實驗目的為建立水楊酸引起自覺性耳鳴之老化促進小鼠(senescence accelerated prone mice; SAMP8)模式,並探討藍綠藻(Spirulina platensis)水萃物,對水楊酸引起耳鳴老化促進小鼠之影響,並以三大方向包括抗氧化、發炎路徑及離子通道基因表現量的改變,來探討藍綠藻萃取物是否能減緩老化動物因水楊酸所引起耳鳴的可能機制。實驗以三月齡的SAMP8小鼠隨機分為三組,分別為控制組、腹腔注射(300mg/Kg BW)水楊酸誘導耳鳴組及管灌(1000mg/Kg BW)藍綠藻水萃物並誘發耳鳴組。實驗為期九天,期間記錄體重與進行主動迴避試驗,動物犧牲後,進行血液生化值GOT、GPT、BUN及Creatinine的分析,並取腦幹(brain stem)、海馬迴(hippocampus)、顳葉(temporal lobe)、額葉(frontal lobe)及內耳(inner ear)進行實驗,抗氧化狀態檢測超氧歧化酶(Superoxide dismutase, SOD)、觸酶(Catalase, CAT)、麩胱甘肽過氧化酶(Glutathione peroxidase, GPx)、血紅素氧化酶-1(Heme oxygenase, HO-1) mRNA表現量,發炎路徑部分測定及血清素2B受體 (5-hydroxytryptamine 2B receptor, 5-HT2B receptor) mRNA表現量,離子通道則測定K+-Cl-聯合轉運體2 (K+-Cl- cotransporter 2, KCC2)及Na+-K+-2Cl-聯合轉運體1(Na+-K+-2Cl- cotransporter 1, NKCC1)的mRNA表現量。由主動迴避試驗結果得知,在成功迴避部分,三組間並無明顯差異;而誘發耳鳴後會使誤判次數增加,藍綠藻水萃物可改善誤判次數,顯示藍綠藻水萃物可以改善水楊酸所引起的自覺性耳鳴。在血液生化值部分GOT、GPT、BUN及Creatinine於三組間皆無顯著差異。水楊酸會抑制各部位的CAT mRNA表現,而藍綠藻水萃物則能增高CAT mRNA表現。與水楊酸組相比,藍綠藻水萃物組可顯著降低各部位SOD mRNA之過度表現。水楊酸組在海馬迴、顳葉、額葉及內耳的KCC2 mRNA有過度表現的現象,但在腦幹三組間並無差異,添加藍綠藻水萃物可顯著減少海馬迴、顳葉、額葉及內耳的KCC2 mRNA之過度表現。而在各部位的GPx、HO-1、NKCC1及5-HT2B receptor 基因表現並無顯著差異。綜合以上結果顯示,藍綠藻水萃物可以改善老化促進小鼠因過量水楊酸所引起自覺性耳鳴,其機制可能與提升抗氧化狀態以及降低KCC2的基因過度表現有關。
Salicylates, also known as aspirin, is known to induce tinnitus. However, the mechanism of generation of tinnitus induced by salicylate is still unclear. Tinnitus is a non-observable phantom sensation, and had no effective treatment until now. Spirulina platensis showed good antioxidant capacity, anti-inflammation and neuroprotective effect. The purpose of this study was to examine the effects of Spirulina platensis on salicylate-induced tinnitus in senescence accelerated prone mice (SAMP8), and evaluated the related gene expression of antioxidative, anti-inflammatory and ion channels. Three-month- old SAMP8 mice were randomly divided into three experimental groups, including normal control, tinnitus group with intrperitoneal injection of salicylate (300 mg/Kg BW), and Spirulina platensis (1000 mg/Kg BW) with salicylate injection tinnitus groups. The active avoidance test were performed during the last nine days of feeding period. After sacrificed, the serum biochemical parameters were analyzed and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and Heme oxygenase-1 (HO-1) 、K+-Cl- cotransporter 2 (KCC2)、Na+-K+-2Cl- cotransporter 1(NKCC1)、5-hydroxytryptamine 2B recrptor (5-HT2B receptor) mRNA expsression of brainstem, hippocampus, temporal lobe, frontal lobe and inner ear were examined. The results showed that correct response score did not significantly different after salicylate injection while the number of false positive response was drastic increase, and spirulina platensis administration could improve the conscious tinnitus. There were not significant differences on GOT, GPT, BUN and creatinine levels among three groups. Salicylate inhibited the CAT mRNA expression and induced the over-expression of SOD mRNA in brain stem, hippocampus, temporal lobe, frontal lobe and inner ear. Spirulina platensis supplementation could increase the CAT mRNA expression and lower the SOD mRNA over-expression when compare with the salicylate only group. Spirulina platensis also could decrease the KCC2 mRNA over-expression in hippocampus, temporal lobe, frontal lobe and inner ear, whereas the difference did not found in the area brain stem. However, there were not significant differences on the GPx , HO-1, NKCC1 and 5-HT2B receptor mRNA expression among three groups. In summary, we concluded that Spirulina platensis could improve the salicylate-induced conscious tinnitus in SAMP8 mice. The mechanism might relate to promote the antioxidantive defense system and reduce the over-expression of KCC2 mRNA.
目錄 頁次
中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖目錄 Ⅶ
表目錄 Ⅸ
縮寫表 Ⅹ
第一章 前言 1
第二章 文獻回顧 2
第一節 水楊酸引起耳鳴之相關性 2
壹 聽覺傳遞路徑 2
貳 耳鳴(Tinnitus)簡介 2
參 耳鳴之原因與機轉 3
圖一 聽覺路徑 3
肆 耳鳴之治療 5
伍 水楊酸簡介 6
圖二 水楊酸化學結構 8
陸 水楊酸與耳鳴之相關性 9
柒 耳鳴動物模式 11
第二節 氧化傷害對老化與疾病之相關性 12
壹 自由基與活性氧物質之介紹 12
貳 自由基與活性氧物質之氧化傷害 13
參 抗氧化防禦系統 14
圖三 哺乳動物細胞中氧化及抗氧化酵素之表現 15
圖四 血紅素氧化酶的反應 18
第三節 血清素及其受體 19
壹 血清素及其受體 19
第四節 氯化物協同轉運體家族(cation-coupled chloride cotransporters,CCCs) 21
壹 K+-Cl-協同轉運體 (K+-Cl- cotransporter, KCC) 22
貳 Na+-K+-2Cl-聯合轉運體(Na+-K+-2Cl- cotransporter, NKCC) 22
第五節 藍綠藻簡介( Spirulina ) 24
第六節 老化促進小鼠(Senescence-accelerated mouse) 29
表一 SAM 之病理生物學表型 30
第三章 材料與方法 31
第一節 實驗動物 31
第二節 耳鳴動物模式建立 31
第三節 實驗流程與方法 32
圖五 實驗流程圖 33
壹、主動迴避試驗(active avoidance test) 34
圖六 主動迴避試驗裝置圖 35
貳 基因表現測定 35
參 PCR產物偵測 37
表二 聚合酶連鎖反應之寡核甘酸引子序列及反應條件 39
第四節 血液生化值測定 41
第五節 統計分析 41
第四章 結果與討論 42
第一節 體重 42
表三 3月齡雄性SAMP8小鼠餵食不同飼料9天後體重之變化 43
第二節 器官重量 44
表四 3月齡雄性SAMP8小鼠餵食不同飼料9天後相對器官重量之比較 45
圖七 3月齡雄性SAMP8小鼠餵食不同飼料9天後腦部不同部位及內耳重量之比較 46
第三節 血液生化值分析 47
表五 3月齡雄性SAMP8餵食不同飼料9天後血液生化值之比較1.2 48
第四節 主動迴避試驗 49
圖八 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後之成功迴避測試比例 51
圖九 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後之誤判次數 52
第五節 抗氧化狀態分析 53
壹 抗氧化酵素之分析 53
圖十 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,腦幹、海馬迴及顳葉之SOD mRNA表現 54
圖十一 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,額葉及內耳之SOD mRNA表現 55
圖十二 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,腦幹、海馬迴及顳葉之CAT mRNA表現 57
圖十三 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,額葉及內耳之CAT mRNA表現 58
圖十四 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,腦幹、海馬迴及顳葉之GPx mRNA表現 60
圖十五 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,額葉及內耳之GPx mRNA表現 61
圖十六 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,腦幹、海馬迴及顳葉之HO-1 mRNA表現 64
圖十七 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,額葉及內耳之HO-1 mRNA表現 65
貳血清素及其受體 66
圖十八 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,腦幹、海馬迴及內耳之5-HT2B receptor mRNA表現 67
參 氯化物協同轉運體家族(cation-coupled chloride cotransporters,CCCs) 68
圖十九 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,腦幹、海馬迴及顳葉之KCC2 mRNA表現 69
圖二十 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,額葉及內耳之KCC2 mRNA表現 70
圖二十一 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,腦幹、海馬迴及顳葉GABAAβ3接受器之表現 71
圖二十二 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,額葉及內耳GABAAβ3接受器之表現 72
圖二十三 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後腦幹、海馬迴及顳葉之NKCC1 mRNA表現 74
圖二十四 3月齡雄性SAMP8小鼠經餵食不同飼料並注射水楊酸後,額葉及內耳之NKCC1 mRNA表現 75
圖二十五、 GABA Aβ3接受器及KCC2 / NKCC1的改變引起耳鳴可能的機制 76
第五章 結論 77
第六章 參考文獻 79
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