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研究生:田宇峯
研究生(外文):Yu-Feng Tian
論文名稱:樟芝前處理對於大白鼠缺血再灌流損傷保護效果之研究
論文名稱(外文):Effects of Antrodia camphorate precondition against ischemia-reperfusion injury of the liver in rats
指導教授:牛柯琪牛柯琪引用關係
指導教授(外文):Ko-Chi Niu
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
學門:生命科學學門
學類:生物科技學類
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:62
中文關鍵詞:肝臟缺血再灌流傷害樟芝凋亡
外文關鍵詞:Ischemia-reperfusion (I/R) injuryAntrodia camphorateapoptosis
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在台灣罹患肝臟疾病的比率越來越高,而慢性肝病和肝硬化近年來更是居十大死亡原因之第六位,此外,肝癌為十大癌症死亡原因之首,肝臟手術及肝臟移植就成了主要治療之方式,肝臟手術及肝臟移植中無可避免地需使用暫時性的肝臟血流阻斷及隨後的再灌流。於臨床上肝臟的缺血再灌流損傷可能發生在肝臟鈍傷、肝臟移植、休克或切除肝臟腫瘤等情況,其詳細機轉至今仍未明。一般認為Kupffer cells的活化誘發氧游離基增加,活化的Kupffer cells亦會釋放出前發炎細胞激素,包括TNF-α及IL-1β,隨後造成靜脈內皮細胞死亡及肝臟細胞之傷害。許多研究顯示肝臟缺血再灌流的損傷,會引起肝臟功能不全或衰竭,而這也成了術後死亡的主因,因此如何提高肝臟手術的安全性以及降低肝臟手術後死亡率及發病率,成了臨床上重要的課題。
樟芝(Antrodia camphorata)又稱為牛樟芝(niu-chang-chih),為台灣特有的一種真菌,只寄生於腐朽中空的牛樟樹(Cinnamomum kanehirai Hayata)上,樟芝是非常重要的中國藥用真菌擔子菌(Basidiomycetes),可用於治療食物及藥物之中毒、腹痛、高血壓及肝癌等疾病。而從樟芝子實體及液態培養之菌絲體萃取出的多醣體已證明具有抗發炎、抗氧化、血管舒張及抗B型肝炎病毒等生理活性。
不同肝臟疾病及肝移植後常會發生缺血再灌流的傷害,目前缺乏有效的方法來預防或治療。本研究建立大鼠肝臟缺血再灌流動物模式,欲以預防的方式給予樟芝,探討其對於大鼠肝臟缺血再灌流傷害是否有保護效果。
本試驗分為缺血再灌流組、樟芝治療組、樟芝sham組及normal control組四組,樟芝治療於缺血前1h 腹腔注射給予0.2 g/kg 樟芝,大鼠麻醉後進行手術,找到肝門靜脈,阻斷肝左葉及中葉的血液流通,使其缺血30分鐘,之後將管線移除恢復灌流6 h或灌流7天觀察存活率。手術過程中進行生理監測,並於缺血前、灌流後2h及6h 檢測血清TNF-α含量。於缺血前、灌流後1h、2h、3h、4h、5h及6h抽血,使用ELSA檢測血清中IL-6及IL-10。灌流後6小時檢測glutamate oxaloacetate transaminase (GOT)、glutamate pyruvate transaminase (GPT)含量,並取其肝臟檢測肝臟組織中malondialdehyde (MDA)含量,並做石蠟包埋切片,分別進行H&E染色及TUNEL染色,進行組織病理學觀察及細胞凋亡觀察。
結果顯示,缺血過程中,以生理監測平均動脈壓的下降做為確認血流阻斷之指標,灌流之後發現血清中TNF-α含量逐漸上升,且灌流後6h肝臟組織有逐漸黑化壞死的現象。樟芝治療確實能降低GPT值,經切片及H&E染色處理也證實樟芝確實能改善肝細胞液泡潰變、血管阻塞及肝組織壞死的現象。生理監測結果顯示,比較樟芝處理組與缺血再灌流組發現對照組體溫明顯高於處理組,樟芝處理組於缺血期間心跳較為穩定,且於灌流後3小時至6小時樟芝處理組平均動脈壓明顯高於缺血再灌流組(P<0.05)。除此之外樟芝治療可降低受缺血再灌流損傷大鼠血清中TNF-α、IL-6含量,以抑制發炎反應。同時IL-10在樟芝治療組中,因發炎反應被抑制而降低上升幅度。樟芝同時也可清除氧化自由基,減少脂質過氧化產物MDA含量,由TUNEL染色結果得知樟芝也可減少肝細胞凋亡的數目,達到保護肝臟的效果。此外灌流後7天發現樟芝能將大鼠存活率提升至80%。
本研究建立大白鼠肝臟缺血再灌流動物模式,且證實樟芝前處理對肝臟缺血再灌流損傷有很好的保護成效,在未來的試驗上,我們將進一步探討其保護的機制並加入其他藥物或治療方法,以達到更好的療效。
It has been found that in recent years the incidence of liver diseases has been increasing greatly in Taiwan. Chronic liver diseases and liver cirrhosis are at the sixth among the top ten causes of death recently. Furthermore, liver cancer is the leading cause of cancer mortality. Liver resection and liver transplantation are the main therapy. Ischemia-reperfusion injury plays an important role during liver resection or liver transplantation. Ischemia-reperfusion (I/R) injury of the liver may occur under many clinical conditions, such as hepatic trauma, hepatic transplantation, hypoperfusion shock or partial hepatectomy for liver tumors. Until now, the actual mechanisms remain unknown. There is evidenced that the sequence of hepatic I/R injury may cause severe liver injury. It was suggested that the increase of Kupffer cells activation induced reaction oxygen species involves the possible mechanisms. Activation of Kupffer cells results in production and release of proinflammatory cytokins, including tumor necrosis factor α (TNF-α) and interleukin 1(IL-1β), chemokines, and neutrophil activation, lead to sinusoid endothelial cell death and hepatic cell damage. The period of hepatic ischemia associated with this technique and the resultant reperfusion can lead to liver injury and dysfunction, which is the main cause of death after hepatectomy. Thus, hepatic I/R injury has been actively investigated, and recently, protective strategies consisting of surgical interventions, pharmacological agents, and gene therapy have been reported. Therefore, enhancing the safety of hepatic surgery and diminishing the significant rates of morbidity and mortality are the most important task in clinical therapy.
In Taiwan, Antrodia camphorate(AC) is an exclusive fungus parasitic on the inner cavity of the endemic species Cinnamomum kanehirai Hayata and an important traditional Chinese medicinal fungus(Basidiomycetes) for the treatment of human disease such as food and drug intoxication, abdominal pain, hypertension and liver cancer. Recently, polysaccharides extracted from fruiting bodies and mycelial cultures of Antrodia camphorate are reported to provide several therapeutic benefits including anti-inflammation, antioxidation, vasorelaxation and anti-hepatitis B virus activities, but the underlying molecular mechanisms are obscure.
Our study had four groups which included ischemia reperfusion group、0.2 g/kg AC + I/R group、AC sham group and normal control group. The I/R rats are treated with 0.2 g/kg Antrodia camphorate (AC) 30 minute before ischemia (IR, ischemia 30min + 0.2 g/kg A.C.), than reperfusion 6h or reperfusion 7 day. Rat serums which were assayed TNF-αquantity by ELISA were extrated before ischemia、after reperfusion 2h and 6h. The serums which were assayed IL-6 and IL-10 quantity by ELISA were extrated before ischemia、after reperfusion 1h, 2h, 3h, 4h, 5h and 6h. After reperfusion 6h the serums were also assayed glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) quantity. Liver tissue【left lateral lobe(ischemia liver) and right lateral lobe(nonischemia liver】will be taken for measuring lipid peroxidation(MDA assay).liver tissue will also be taken for paraffin-embedded tissue sections which were respectively stained by H&E or TUNEL, and observed histopathology and liver cell apoptosis.
In these results, the ischemia reperfusion animal modal was successful,when MBP decreased at ischemia stage and increased gradually at reperfusion stage. Furthermore the liver was black and necrosis during reperfusion 6h. Pretreated Antrodia camphorate, we detected that rat GPT concentration was improved at reperfusion 6h stage, contrasted with IR control (P<0.05), but GOT concentration was decreased slighter in pretreated Antrodia camphorate. The paraffin-emmbedded tissue sections which were stained by H&E showed that AC could diminish hepatocyte vacuolar degeneration, vascular congestion and less severe hepatic necrosis in liver grafts at reperfusion 6 hours. The physiology monitory result showed : 1. After ischemia injury, AC could avoid high body temperature. 2. At ischemia stage, AC could maintain heart rate stability. 3. Contrasted with IR control, AC could maintain blood pressure stability at reperfusion 3~6 h. Under other hand, AC could reduce the TNF-α、IL-6 quantity of liver ischemia reperfusion injury rat serum, and suppress inflammation reaction. In AC therapy group, because inflammation reaction were be suppressed, IL-10 quantity range on the rise were be down regulating in serum. When we assayed MDA level., which was decreased at Antrodia camphorate pretreatment, compared with IR control after reperfusion 6h. In the result of TUNEL stain, AC reduced severe hapatic cell DNA fragmentation and apoptosis in liver at reperfusion 6 hours. At reperfusion 7 day, we found AC which could increase the survival rate to 80%。
In this study, we had established rat liver ischemia reperfusion modal. The results suggested, effect of Antrodia camphorate precondition to protect was good, which could reduce liver ischemia reperfusion injury. In the future, we will investigate the mechanism of protection with Antrodia camphorate, and add other medicine or therapy method which can increase the curative effect of liver ischemia reperfusion injury.
書名頁 i
博碩士論文授權書 ii
論文口試委員審定書 iii
摘要 iv
誌謝 ix
目錄 x
圖目錄 xii
第一章 文獻回顧 1
1.1 缺血再灌流對臨床之重要性 1
1.2 缺血再灌流傷害特性 3
1.3 缺血再灌流傷害中氧自由基所扮演之角色 4
1.4缺血再灌流傷害中細胞激素與化學激素所扮演之角色 7
1.5缺血再灌流損傷的防禦機制 9
1.6牛樟菇應用於臨床疾病 10
1.7本實驗目的 12
第二章 研究方法 13
2.1實驗動物 13
2.2實驗設計 14
2.3實驗分組 15
2.4實驗方法 16
2.4.1 動物手術及生理參數監測 16
2.4.2 肝臟缺血再灌流模式的建立 16
2.4.3 樟芝之配置 17
2.4.4 檢測TNF-α、IL-6、IL-10活性 17
2.4.5 檢測脂質過氧化 18
2.4.6 組織病理學檢測 18
2.4.7 細胞凋亡檢測 18
2.4.8 樟芝治療肝臟缺血再灌流存活率試驗 19
2.5 統計學分析 19


第三章 實驗結果 20
3.1建立大鼠肝臟缺血灌流動物模式 20
3.2給予樟芝前處理後進行肝臟缺血再灌流期間之生理監測 21
3.3給予樟芝前處理後進行肝臟缺血再灌流期間GOT、GPT之變化及組織病理學檢測 22
3.4給予樟芝前理後進行肝臟缺血再灌流期間,大鼠血清中TNF-α、IL-6、IL-10含量之表現 23
3.5肝組織MDA含量分析 24
3.6肝組織細胞凋亡分析 25
3.7 存活率試驗 26
第四章 討論 27
4.1大鼠動物模式的建立 27
4.2樟芝對缺血再灌流大鼠肝指數及生理參數的影響 28
4.3樟芝對缺血再灌流大鼠發炎反應因子的影響 29
4.4腹腔注射樟芝抗氧化能力探討 30
4.5樟芝對缺血再灌流大鼠肝組織細胞凋亡的影響 30
4.6腹腔注射樟芝毒性探討 31
4.7樟芝治療肝臟缺血再灌流存活率試驗 31
第五章 結論 32
參考文獻 33
圖1 肝臟缺血再灌流免疫反應路徑圖 40
圖2 缺血再灌流免疫反應流程 41
圖3 缺血誘發過氧化路徑圖 42
圖4 缺血再灌流手術策略 43
圖5 實驗流程圖 44
圖6 實驗設計圖 44
圖7 大白鼠的保定及肝臟缺血再灌流之手術過程 45
圖8 大白鼠之肝臟,可分為右外側葉,左外側葉,中葉以及前葉 46
圖9 大白鼠肝臟缺血再灌流左外側葉及中葉手術之技術 47
圖10 肝臟缺血再灌流模式建立手術過程紀錄照 48
圖11肝臟缺血再灌流動物模式平均動脈壓監測圖 49
圖12給予樟芝後進行肝臟缺血再灌流生理監測圖 51
圖13大鼠缺血再灌流血清GOT、GPT含量 52
圖14大鼠肝臟缺血再灌流肝臟組織H&E染色 54
圖15大鼠肝臟缺血再灌流檢測血清中TNF-α含量 55
圖16肝臟缺血再灌流檢測血清中IL- 6含量 56
圖17肝臟缺血再灌流檢測血清中IL- 10含量 57
圖18大鼠肝臟缺血再灌流肝臟組織MDA含量 58
圖19大鼠肝臟缺血再灌流肝組織細胞凋亡TUNEL分析 60
圖20大鼠肝臟缺血再灌流存活率試驗 61
圖21樟芝保護肝臟缺血再灌流路徑圖 62
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