臺灣博碩士論文加值系統

1.研究背景：
糖尿病是現代社會中一項重要的疾病，它會造成全身嚴重的併發症。糖尿病不論在先進國家或開發中國家都造成很高的罹病率與死亡率。而糖尿病視網膜病變是造成後天性人口失明最常見的原因。糖尿病視網膜病變是一項終會導致失明的嚴重疾病，它與視網膜血管新生極度相關。一般認為高血糖、advanced glycation end-products(AGEs)和網膜缺氧(hypoxia)是造成視網膜血管新生的主因。
氧化壓力(oxidative stress)被認為是導致糖尿病視網膜病變的關鍵所在。所謂氧化壓力指的是氧化劑(包括reactive oxygen species (ROS)和 reactive nitrogen species (RNS))及抗氧化劑之間嚴重的失衡。慢性的氧化壓力會造成許多生物分子的損傷，如DNA、脂質、蛋白質和碳水化合物。視網膜有大量的不飽和脂肪酸以及比其他組織更高的氧氣攝取量與葡萄糖代謝率，所以更容易遭受氧化壓力的傷害。糖尿病性視網膜病變除了氧化壓力的影響之外，與慢性發炎症狀也有許多的關聯。發炎及許多引起發炎的因子(cytokines)在糖尿病視網膜病變的產生及進展上，扮演十分重要的角色。
蝦紅素Astaxanthin（AST），是一種強大的生物抗氧化劑。由於蝦紅素具有良好的抗氧化作用及抗發炎作用的功能，我們假設AST可能減少減少氧化介質的產生，並增加抗氧化防禦系統的功能。 除此之外，蝦紅素也可以也減少發炎介質的產生。
2.研究目的
本研究是為了評估蝦紅素(AST)在streptozotocin誘發之糖尿病大鼠模式下，對於這些代表氧化壓力的生化指標及發炎媒介物表現程度的影響。希望能證實蝦紅素在糖尿病視網膜病變中能有抗發炎及抗氧化的效果，並建立起蝦紅素應用於預防糖尿病視網膜病變的模式。
3.研究方法
本研究採用50隻50至60天大的Wistar雌性實驗大鼠，體重介於 200~250g。再將其分為五組：(1)健康對照組 (2)糖尿病組 (3)糖尿病+低劑量Astaxanthin組 (4)糖尿病+高劑量Astaxanthin組 (5) 糖尿病+Lutein組。40隻糖尿病大鼠再任意分為四組：10隻低劑量Astaxanthin組給予兩個月的胃內灌注astaxanthin (0.6 mg/kg/day)；10隻高劑量Astaxanthin組給予兩個月的胃內灌注astaxanthin (3 mg/kg/day)；10隻Lutein組給予兩個月的胃內灌注lutein (0.5 mg/kg/day)；其餘10隻給予兩個月的胃內灌注等量的0.9%生理食鹽水。兩個月後，總共49隻大鼠給予腹腔內注射致死劑量的pentobarbital。然後，每隻老鼠的眼球迅速地被取下以取得新鮮的視網膜及房水組織檢體來作研究。
4.結果
在氧化壓力媒介物的表現程度上，藉由免疫組織化學染色法(IHC)去研究。可以發現因過度氧化而產生的8OH-dG、nitrotyrosine 及acrolein，在蝦紅素實驗組糖尿病大鼠中，視網膜的含量都有減少的趨勢。在抗氧化系統上，藉由RT-PCR方法去研究，蝦紅素也可以成功增加抗氧化酶(heme oxygenase、Thioredoxins、Peroxiredoxin I )的表現。
在發炎媒介物的表現程度上，藉由IHC染色、西方點墨法、ELISA及PCR實驗方法去研究。蝦紅素可以成功降低多項和糖尿病視網膜病變相關的發炎媒介物質，它會抑制發炎作用而減少 ICAM-1、MCP-1 及FKN蛋白質在前房、玻璃體及視網膜上的濃度，也會減少ICAM-1、FKN、IL-8、RANTES的mRNA在視網膜的表現。
根據 IHC 和 EMSA 的結果，蝦紅素同樣地也抑制了NF-κB在視網膜中的活性。
5.結論
本實驗動物模式證實蝦紅素的確有保護糖尿病視網膜的功效，會減少在大鼠之糖尿病視網膜病變中的氧化壓力及發炎作用。希望在未來，可以嘗試用更多不同濃度的蝦紅素來進行更廣泛的動物實驗、細胞組織培養、及人體實驗。

1.Background
Diabetic retinopathy, a sight-threatening complication of diabetes, is the major cause of blindness in young adults. Studies have documented that sustained hyperglycemia is the instigating cause of disrupted normal cellular metabolism leading to the development of retinopathy. Diabetes increases oxidative stress, and increased oxidative stress is one of the key regulators in the development of diabetic complications.
Oxidative stress results from an imbalance between oxidant production, including reactive oxygen species (ROS), reactive nitrogen species (RNS), and antioxidant defense mechanisms.The outer retina is avaseular and receives its oxygen supply from the choroid,which lacks hyperoxia-induced autoregulation. Therefore, photoreceptors are exposed to higher level of tissue oxygen than most other tissue. This phenomenon renders retina more susceptible to oxidative stress.Consequences of chronic oxidative stress include damage to biological macromolecules such as DNA, lipids, proteins, and carbohydrates.
An association between oxidative stress and the development of diabetes complications has been recognized for over 20 years.Increased production of reactive oxygen species has been strongly implicated in the pathogenesis of diabetic retinopathy. Besides, inflammation may also play a central role in the development and progression of diabetic retinopathy.
Astaxanthin (AST), a dietary carotenoid, is often decreasing the formation of products of oxidative damage induced by biological molecules.Astaxanthin is a powerful biological antioxidant occurring naturally in a wide variety of living organisms, and is present in many well-known sea foods such as salmon, trout, red sea-bream, shrimp, lobster and fish eggs. Recent studies have shown that astaxanthin is a powerful biological antioxidant. Due to potent anti-oxidative and anti-inflammatory effects of AST, we hypothesize that AST could reduce the oxidative mediators and increase antioxidant enzyme defense system. Beside, AST may also reduce inflammatory mediators.
2.Purpose
This study is to evaluate the potential protective effects of AST against retinal oxidative damage in streptozotocin(STZ) - induced diabetic rats.
3.Material and Methods
Fifty 6 weeks old female Wistar rats with the body weight about 200-250g were divided into a healthy control group(n=10) and a STZ- induced diabetic group(n=40). These forty rats received intra- peritoneal injection with STZ to induce diabetes and randomly divided into four groups. A group of rats received normal powdered diet or powdered diet supplemented with 0.6 or 3mg/kg AST or 0.5mg/kg lutein.
Eight weeks later, the eyeball of each rat was taken out to obtain fresh retina tissues. We performed immunohistochemistry (IHC), western blot analysis, enzyme-linked immunosorbent assay (ELISA),reverse transcription - polymerase chain reaction (RT-PCR), and electrophoretic mobility shift assays (EMSA) to assess oxidative stress and inflammatory mediators in diabetic retinopathy.
4.Results and discussions
AST could reduce the oxidative mediators (8-hydroxy-2''- deoxyguanosine (8-OHdG), Nitrotyrosine ,and acrolein ) and increase antioxidant enzyme defense system(heme oxygenase, peroxiredoxin, and thioredoxin ). AST also reduce inflammatory mediators (intercellular adhesion molecule (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fractalkine (FKN)) and the activity of transcription factor NF-κB.
5.Conclusions
Astaxanthin could reduce oxidative stress and inflammatory reactions in diabetic retina rat model .The antioxidant and anti-inflammatory effect of Astaxanthin was supposed to be mediated by inhibition of NF-κB activity and reduced the downstream products. Astaxanthin acted as antioxidants by facilitating the activity of antioxidant enzymes to inhibit oxidative stress and reduced the damage to biological macromolecules such as DNA, lipids and proteins. Axtaxanthin seems to be the nutritional supplement for prevention of diabetic retinopathy progression.

口試委員會審定書………………………………………………………i
誌謝…………………………………………………………………… ii
中文摘要………………………………………………………………iii
英文摘要……………………………………………………………… vi
第一章 緒論……………………………………………………………1
1.1 糖尿病視網膜病變簡介……………………………………1
1.2 糖尿病視網膜病變的臨床分期治療方法…………………1
1.3 糖尿病視網膜病變的致病機轉……………………………3
1.4 氧化壓力與糖尿病視網膜病變……………………………5
1.5 發炎反應與糖尿病視網膜病變……………………………6
1.6 抗氧化劑與糖尿病視網膜病變……………………………7
1.7 Astaxanthin之功效………………………………………8
1.8 Astaxanthin與糖尿病……………………………………9
第二章 研究假說及研究目的…………………………………………10
2.1 研究假說…………………………………………………10
2.2 研究目的…………………………………………………11
第三章 研究方法及材料………………………………………………12
3.1 動物實驗模式……………………………………………12
3.2 免疫組織化學染色法Immunohistochemistry…………13
3.3 西方墨點轉漬法 Western blotting……………………13
3.4 酵素連結免疫分析法Enzyme-linked immunosorbent assay………… 14
3.5 備製RNA、cDNA及聚合酶連鎖反應Polymerase Chain Reaction…… 14
3.6 電泳遷移率檢測 Electrophoretic Mobility Shift Assays………… 15
3.7 資料收集及統計分析……………………………………16
第四章 研究結果………………………………………………………17
4.1 大鼠基本資料：體重及血糖……………………………17
4.2 免疫組織化學染色法結果………………………………17
4.3 西方墨點轉漬法結果……………………………………18
4.4 酵素連結免疫分析法結果………………………………19
4.5 聚合酶連鎖反應結果……………………………………19
4.6 電泳遷移率檢測結果……………………………………20
第五章 討論…………………………………………………………21
5.1 糖尿病視網膜病變與氧化壓力之相關性……………21
5.2 糖尿病視網膜病變與慢性發炎反應之相關性…………22
5.3 Astaxanthin的抗氧化及抗發炎作用…………………22
第六章 未來展望………………………………………………………24
第七章 參考文獻………………………………………………………26
圖目錄
圖一、糖尿病視網膜病變機轉………………………………………34
圖二、蝦紅素(astaxanthin)的化學結構……………………………35
圖三、本實驗研究假說示意圖………………………………………36
圖四、實驗動物分組及實驗研究流程圖……………………………37
圖五、大鼠視網膜切片的H&E 染色和免疫組織染色negative control………38
圖六、氧化壓力之過氧化物質指標(8-OHdG、Nytrotyrosin、Acrolein)免疫組織化學染色…………………………………………39
圖七、過氧化物質指標免疫組織化學染色成像之光學濃度定量… 40
圖八、發炎反應指標ICAM-1、MCP-1及FKN免疫組織化學染色………41
圖九、發炎反應指標免疫組織化學染色成像之光學濃度定量………42
圖十、大鼠視網膜 NF-κB p65免疫組織化學染色及光學濃度定量 43
圖十一、西方墨點轉漬法結果…………………………………………44
圖十二、酵素連結免疫分析法結果……………………………………45
圖十三、PCR結果：大鼠視網膜抗氧化防禦酵素mRNA之表現……… 46
圖十四、PCR結果：大鼠視網膜MCP-1、 ICAM-1及FKN mRNA之表現 47
圖十五、EMSA結果：大鼠視網膜NF-κB p65之活性…………………48
表目錄
表一、西方墨點轉漬法第一級及第二級抗體…………………………49
表二、聚合酶連鎖反應引子之相關資訊………………………………50
表三、大鼠體重及血糖基本資料………………………………………51

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