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研究生:黃冠婷
研究生(外文):Guan-Ting Huang
論文名稱:D-甲硫胺酸改善順鉑誘導大鼠心臟毒性以及厭食之探討
論文名稱(外文):Improvement of Cisplatin-induced Cardiotoxicity and Anorexia by D-methionine in Rats
指導教授:歐珠琴
指導教授(外文):Chu-Chyn Ou
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:營養學系碩士班
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:2017
畢業學年度:105
語文別:中文
論文頁數:117
中文關鍵詞:D型甲硫胺酸順鉑厭食心臟毒性
外文關鍵詞:D- methioninecisplatinanorexiacardiotoxicity
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順鉑 (Cisplatin) 是一種含鉑且常見的化學治療藥物,由於cisplatin會產生嚴重的副作用,如:耳毒性、腎毒性、神經毒性、心臟毒性、厭食/惡病質及腸胃道毒性等。D型甲硫胺酸 (D-methionine) 是L型甲硫胺酸的右旋異構物,目前已知藉由其抗氧化特性,降低cisplatin所引起的耳毒性與腎毒性,然而,D-methionine對於cisplatin引起的厭食與心臟毒性是否具有保護作用,尚未被探討,本實驗藉由大鼠模式探討D-methionine是否可改善cispaltin所誘導的厭食與心臟毒性。我們將Wistar品系雄性大鼠隨機分成四組,每組5-8隻,其中,控制組 (Control) 與順鉑組 (Cisplatin) 在實驗的前3天至第18天,管餵一般飲用水,且分別在實驗的第1、8、15天進行腹腔注射0.9%NaCl與cisplatin,劑量皆為5 mg/kg;順鉑合併D型甲硫胺酸組 (Cisplatin+D-methionine) 與D型甲硫胺酸組 (D-methionine) 分別在注射第一劑cisplatin與0.9%NaCl的前三天,開始灌食D-methionine 300 mg/kg直到實驗的第18天,並分別在實驗的第1、8、15天進行腹腔注射cisplatin與0.9%NaCl,劑量皆為5 mg/kg,最後在實驗的第19天進行犧牲。我們檢測與食慾調節相關的因子 (例如:飢餓素、瘦體素、色胺酸羥化酶1、5-HT3 receptor、5-HT2C receptor、NPY、AgRP、POMC、CART) ,藉由生理紀錄器測量心跳、收縮壓、舒張壓、平均動脈壓、心電圖,以及心臟毒性指標 (CK-MB、LDH) 與發炎反應 (IL-6、IL-1β、TNF-α) 、氧化壓力 (MDA、GSH、GPx、SOD) ,作為心臟毒性指標檢測。研究結果顯示,在厭食方面,D-methionine可以顯著改善cisplatin誘導的體重與攝食量降低,減少胃內容物殘留量並增加胃排空,透過降低腸道中色胺酸羥化酶1 (TPH1) 的活性、5- HT3 receptor的陽性反應以及降低下視丘中5-HT2C receptor基因表現等改善胃腸功能失調進而減緩厭食的發生;此外,D-methionine透過增加血清中瘦體素的含量,而促進下視丘中POMC與CART的基因表現,與降低NPY與AgRP的基因表現,以維持能量的平衡狀態。在心臟毒性方面,D-methionine可以輕微改善心臟功能異常,包括心跳、收縮壓、舒張壓以及平均動脈壓,但是無法改善cisplatin所引起的心臟毒性 (LDH與CK-MB) 與發炎反應 (IL-6、IL-1β、TNF-α) ;我們也發現cisplatin並不會造成心律不整、胸主動脈受損、心臟組織損傷以及氧化壓力。
因此,口服D-methionine具有改善cisplatin所引起厭食的潛力,但無法減緩心臟毒性。
Cisplatin, a platinum-containing compound is one of the most common chemotherapeutic drugs and leads to many serious side effects, such as ototoxicity, nephrotoxicity, neurotoxicity, cardiotoxicity, anorexia/cachexia and gastrointestinal toxicity. D-methionine, a dextral isomer of L-methionine is a sulfur-containing antioxidant. Owing to its antioxidant properties, D-methionine may attenuate ototoxicity and nephrotoxicity caused by cisplatin. However, effects of D-methionine on cisplatin-induced anorexia and cardiotoxicity have not been explored yet. In this study, we investigated whether D-methionine could attenuate cisplatin-induced anorexia and cardiotoxicity in rat model. Male Wistar rats were randomly divided into four groups of which from five to eight rats each. The control group and cisplatin group were administrated water from three days before to the 18th day of the experiment and injected with 0.9% NaCl and cisplatin (5 mg/kg) via intraperitoneal (i.p.) on the 1st, 8th and 15th day of the experiment respectively. Cisplatin plus D-methionine group and D-methionine alone group were pre-administrated daily with D-methionine (300 mg/kg) 3 day before cisplatin and 0.9% NaCl treatment until the study is completed. Also cisplatin and 0.9% NaCl were i.p. injected on the 1st, 8th and 15th day of the experiment, respectively. All animals were sacrificed on day 19 of the experiment. We detected the appetite regulated factors such as ghrelin, leptin, tryptophan hydroxylase 1, 5-HT3 receptor, 5-HT2C receptor, NPY, AgRP, POMC, CART, and used physiological recording machine to determine heart rate, systolic blood pressure, diastolic blood pressure, mean arterial pressure and electrocardiogram. The cardiotoxicity marker (CK-MB, LDH) ,inflammatory response (IL-6, IL-1β, TNF-α) and oxidative stress (MDA, GSH, GPx, SOD) were measured and deemed as cardiotoxicity index. As for anorexia, the results showed that D-methionine can significantly improve cisplatin-induced weight loss and food intake, decrease gastric contents, enhance gastric emptying in the stomach tissue. D-methionine could reduce the activity of tryptophan hydroxylase 1 (TPH1), increase the positive response of 5-HT3 receptor in the intestine and decrease gene expression of 5-HT2C receptor in the hypothalamus in cisplatin-treated rats. In addition, to maintain energy balance in cisplatin-treated rats, D-methionine increased plasma levels of leptin in serum, which lead to enhance the gene expression of POMC, CART and decrease the gene expression of NPY and AgRP. By the results of cisplatin-induced cardiotoxicity, we found D-methionine can slightly improve the abnormal status of heart rate, systolic blood pressure, diastolic blood pressure, and mean arterial pressure, while it cannot ameliorate LDH and CK-MB activity and inflammatory response (IL-6 , IL-1β, TNF-α) after cisplatin injection. Besides, cisplatin did not cause arrhythmia, thoracic aorta damage, cardiac tissue damage and oxidative stress. These results indicate that oral D-methionine may have potential for improving cisplatin-induced anorexia but can not ameliorate cisplatin induced cardiotoxicity.
中文摘要
英文摘要
對照縮寫表
第一章 文獻探討 1
第一節 順鉑 (Cisplatin) 1
第二節 癌症厭食症 (Cancer anorexia) 4
第三節 心臟毒性 (Cardiotoxicity) 12
第四節 D型甲硫胺酸 (D-methionine) 15
第二章 研究動機 20
第三章 材料方法 21
第一節 設備與藥品 21
第二節 動物實驗設計 25
第三節 實驗方法 27
一、 測量心跳 (Heart rate) 、血壓 (Blood pressure) 、心電圖(Electrocardiogram) 27
二、 血液收集及分析 28
三、 臟器收集 28
四、 胃排空率計算 28
五、 蘇木精 — 伊紅染色法 (Hematoxylin-Eosin staining, H&E stain) 29
六、 三色染色 (Masson’s trichrome staining, MT stain) 29
七、 免疫組織化學染色法 (immumohistochemistry staining, IHC stain) 30
八、 乳酸脫氫酶 (Lactate dehydrogenase, LDH) 測定 31
九、 MB型肌酸酐酶 (Creatine kinase-myocardial band, CK-MB) 31
十、 反轉錄酶聚合連鎖反應 (Reverse transcription-polymerase chain reaction, RT-PCR) 32
十一、 凝膠洋菜瓊脂 (Agarose gel ) 電泳分析 35
十二、 組織臟器樣品製備 36
十三、 總蛋白質 (Total protein) 濃度測定 37
十四、 麩胱甘肽 (Glutathione, GSH) 含量測定 38
十五、 麩胱甘肽過氧化酶 (Glutathione peroxidase, GPx) 活性測定 39
十六、 超氧岐化酶 (Superoxide dismutase, SOD) 活性測定 41
十七、 丙二醛 (Malondialdehyde, MDA) 含量測定 42
十八、 細胞激素 (Cytokine:IL-6、IL-1β、TNF-α) 含量測定 43
十九、 色胺酸羥化酶1 (Tryptophan hydroxylase 1,TPH1) 活性測定 45
二十、 統計方法 47
第四章 實驗結果 48
第一節 探討D-methionine改善cisplatin誘導大鼠的厭食/惡病質 48
一、 D-methionine改善cisplatin誘導的體重下降 48
二、 D-methionine改善cisplatin誘導的攝食量降低 49
三、 觀察D-methionine影響cisplatin誘導的各器官重量變化 50
四、 D-methionine改善cisplatin誘導的脂肪與腿部肌肉質量減少 50
五、 D-methionine改善cisplatin誘導的胃排空障礙 51
六、 探討D-methionine影響cisplatin誘導的飢餓素變化 52
七、 探討D-methionine對cisplatin影響血清素的變化 52
八、 探討D-methionine對cisplatin影響的瘦體素的變化 54
九、 探討D-methionine對cisplatin影響的食慾相關神經元胜肽 54
第二節 探討D-methionine改善cisplatin誘導大鼠的心臟毒性 56
一、 D-methionine改善cisplatin誘導的心臟功能 56
二、 觀察D-methionine影響cisplatin誘導的胸主動脈型態變化 ……………………………………………………………..57
三、 D-methionine對cisplatin誘導的心臟毒性 57
四、 D-methionine對cisplatin誘導的氧化傷害與抗氧化能力 ……………………………………………………………..58
五、 D-methionine影響Cisplatin誘導的心臟發炎 59
六、 觀察D-methionine影響cisplatin誘導的心臟組織型態變化………………… 59
第五章 討論 60
第六章 圖表 76
第七章 附錄 99
第八章 參考文獻 102
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