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系統識別號 U0007-0308201114053800
論文名稱(中文) 四氯化碳誘導肝臟發炎動物模式中D-乳酸之探討
論文名稱(英文) The Study of D-Lactate in Carbon Tetrachloride-Induced Liver Inflammation
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 99
學期 2
出版年 100
研究生(中文) 莊明正
研究生(英文) Ming-Cheng Chuang
學號 M301098004
學位類別 碩士
語文別 中文
口試日期 2011-07-11
論文頁數 67頁
口試委員 委員-李憶菁
委員-陳福安
委員-陳世銘
指導教授-林本元
共同指導教授-李仁愛
中文關鍵字 四氯化碳  D-乳酸  甲基乙二醛  NBD-PZ  5,6-二胺基-2,4-二羥基-嘧啶硫酸鹽(DDP)  化學性肝病變 
英文關鍵字 Carbon tetrachloride  D-lactate  methylglyoxal  NBD-PZ  5,6-diamino-2,4-hydroxypyrimidine sulfate (DDP)  chemical hepatopathy 
學科別分類
中文摘要 D-lactate目前已經被證實和許多疾病有相關性,如糖尿病、短腸症、菌血症等,而本實驗室先前的研究也發現,在各種不同的腎臟損傷下,如糖尿病慢性腎損傷,馬兜鈴酸急性腎損傷等,尿液中D-lactate有明顯上升的現象,顯示尿中D-lactate可能可以作為腎臟損傷的指標;然而此現象在其他器官組織受損時是否也會出現,目前仍然是有待研究的。Ruan等人發現在肝硬化病人身上,血清中D-lactate有上升的現象,然而至於尿中和組織中之D-lactate是否也有上升的現象,目前顯少有研究討論;此外,D-lactate之前趨物─甲基乙二醛(Methylglyoxal;MG)之變化和組織受損間關係的研究目前也相對缺乏。因此,為了探討生物體內D-lactate和MG與肝臟發炎之關係,本研究選用四氯化碳誘導肝臟發炎的動物模式,以觀察生物體內D-lactate、MG與肝臟發炎的關係。
實驗動物為雄性Wistar大鼠,以腹腔注射方式給予0.3 mL / kg體重/ 週 之四氯化碳(溶於大豆油),共給予四週以誘導其肝臟發炎,以組織病理切片及血清中AST和ALT以作為肝臟發炎之指標,並同時分別觀察尿液、血清和肝臟組織中D-lactate、MG之含量。結果發現,接受四氯化碳投予之大鼠組織切片中有明顯慢性發炎和纖維化的現象,而此時期肝臟組織中MG的量也明顯增加(與均質液蛋白質之比分別為0.1282 ± 0.0188 vs. 0.2481 ± 0.0649 µg/mg, p = 0.004),而血清、尿液以及肝臟組織中D-lactate在此時期也都有明顯上升的現象(57.65 ± 2.59 vs. 92.16 ± 16.69 µM, p = 0.003;1.056 ± 0.071 vs. 1.555 ± 0.366 µmol / mg Ucr, p = 0.017;0.8598 ± 0.0402 vs. 1.2933 ± 0.0782 nmol / mg protein, p < 0.001)。
本研究證實,在四氯化碳誘導肝臟發炎下,肝臟組織中MG的含量會 明顯增加;而在尿液、血清和組織中D-lactate也有明顯上升,顯示MG及D-lactate和四氯化碳所造成之肝臟發炎有一定之相關性。
英文摘要 D-lactate has been found related to several diseases, diabetes mellitus, short bowel syndrome and bacteremia, etc for instance. In previous studies by our lab, urinary D-lactate level elevated significantly under numerous renal injuries, such as diabetes nephropathy and aristolochic acid-induced acute renal injuries, which indicated that urinary D-lactate level might be used as a biomarker for renal injuries. However, would such circumstances happen in other organ injuries or not remains unclear. Ruan et al found that in patients with liver cirrhosis, serum D-lactate increased significantly. Nevertheless, few study talks about D-lactate in urine and tissues. Moreover, the relationship between tissue injuries and methylglyoxal, the precursor of D-lactate, is still not well-established. Carbon tetrachloride- induced liver inflammation animal model was chosen in order to investigate the relationship of among D-lactate, Methylglyoxal and liver inflammation.
Male Wistar rat was used in this study with intraperitoneal injection of carbon tetrachloride in a dose of 0.3 mL / kg body weight / week (Dissolved in soybean oil). Histological examination and serum liver enzymes were used as the biomarker of liver inflammation. The results showed that chronic inflammation and fibrosis were found in carbon tetrachloride-treated rats. Meanwhile, methylglyoxal level in liver tissue increased significantly (0.1282 ± 0.0188 vs. 0.2481 ± 0.0649 µg/mg corrected with protein concentration, p = 0.004). D-lactate levels in serum, urine and liver tissue also appear significant trends of elevation.
This study demonstrated that under such situation of carbon tetrachloride-induced liver inflammation, methylglyoxal level in liver tissue and D-lactate level in serum, urine and liver tissue increased significantly which indicated that methylglyoxal and D-lactate are related to carbon tetrachloride-induced liver inflammation.
論文目次 目錄 ……………………………………………………………………………….. I
附圖目錄 ………………………………………………………………………….. IV
附表目錄 ………………………………………………………………………….. VI
縮寫表 …………………………………………………………………………….. VII
中文摘要 ………………………………………………………………………….. VIII
英文摘要 ……………………………………………………………………………. .X

第一章 緒論
第一節 前言 ……………………………………………………………………… 1
第二節 乳酸 ……………………………………………………………………… 4
1.2.1 乳酸簡介 ………………………………………………………………. 4
1.2.2 生物體中D-Lactate的來源 ……………………………………………. ..5
1.2.3 Lactate之分佈與代謝 ………………………………………………… .5
1.2.4 D-Lactate之相關疾病 ………………………………………………… .6
第三節 甲基乙二醛(Methylglyoxal) ………………………………………... 9
第四節 四氯化碳誘導肝臟病變之機轉 ………………………………………… 12
第五節 研究目的 ………………………………………………………………… 15
第二章 實驗材料與方法
第一節 實驗動物及材料 ………………………………………………………… 16
2.1.1 實驗動物 ………………………………………………………………. 16
2.1.2 實驗試藥 ………………………………………………………………. 16
2.1.3 實驗儀器 ………………………………………………………………. 18


第二節 實驗方法 …………………………………………………………………… ..20
2.2.1 分析方法 ………………………………………………………………. 20
2.2.1.1 D-Lactate之分析方法 ……………………………………………. .20
2.2.1.2 Methylglyoxal之分析方法 ………………………………………. .23
2.2.2 分析方法之確效 ……………………………………………………… .25
2.2.2.1 Methylglyoxal分析方法之確效 …………………………………. .25
2.2.3 四氯化碳誘導肝臟損傷實驗模式 …………………………………… .26
2.2.3.1 實驗動物品系及四氯化碳投與劑量 ……………………………... .26
2.2.3.2 四氯化碳投與方法與實驗設計 ………………………………….. 26
2.2.3.3 動物犧牲法、組織切片製作與Hematoxylin-Eosin Stain …….. 28
2.2.4 D-Lactate之分析 …………………………………………………..... 28
2.2.4.1 尿液中D-Lactate分析 ……………………………………………….. 28
2.2.4.2 血清中D-Lactate分析 ……………………………………………… 29
2.2.4.3 肝臟組織中D-Lactate分析 ………………………………………… 29
2.2.5 Methylglyoxal之分析 ……………………………………………….. 30
2.2.5.1 尿液中Methylglyoxal分析 ………………………………………….. .30
2.2.5.2 血清中Methylglyoxal分析 ………………………………………… 30
2.2.5.3 肝臟組織中Methylglyoxal分析 …………………………………… 30
2.2.6 蛋白質定量 …………………………………………………………….. 31
2.2.7 血液及尿液生化指標分析 …………………………………………….. 31
第三節 統計分析方法 ……………………………………………………………. 33
第三章 實驗結果
第一節 分析方法之確效 ………………………………………………………… 34
3.1.1 Methylglyoxal分析方法之確效試驗 ………………………………… 34
3.1.1.1檢量線線性(Linearity)…………………………………………….. 34
3.1.1.2準確度(Accuracy)和精密度(Precision)……………………… 34
3.1.2 D-Lactate分析方法之確效試驗 ……………………………………… 38
第二節 大鼠四氯化碳肝臟發炎模式之建立 ……………………………………. 41
3.2.1 血液生化指標之變化 ………………………………………………….. 41
3.2.2 生物檢品中D-Lactate含量 ……..……………………………………. 43
3.2.2.1 尿中D-Lactate含量 ………………………………………………… 43
3.2.2.2 血清中D-Lactate含量 ……………………………………………… 45
3.2.2.3 肝臟組織中D-Lactate含量 ………………………………………… 47
3.2.3 生物檢品中Methylglyoxal含量 ……………………………………... 49
3.2.3.1尿中Methylglyoxal含量 ……………………………………............ 49
3.2.3.2血清中Methylglyoxal含量 ………………………………………….. ..51
3.2.3.3肝臟組織中Methylglyoxal含量 …………………………………….. ..53
3.2.4 組織病理切片H&E染色 ………………………………………………. .55
第四章 討論 ………………………………………………………………………. ..58
第一節 大鼠四氯化碳誘導肝臟發炎模式之建立 ……………………………….. .58
第二節 MG、D-Lactate與CCl4誘導肝臟發炎之關係 ……………………….. .61
第五章 結論 ………………………………………………………………………. ..64
參考文獻 ……………………………………………………………………………... ..65
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