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研究生:鄭士豪
論文名稱:蟬花發酵產物對 Cisplatin 誘導 SD 大鼠腎損傷的保護作用
指導教授:邱駿紘邱駿紘引用關係喬長誠喬長誠引用關係
學位類別:碩士
校院名稱:弘光科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:100
語文別:中文
論文頁數:126
中文關鍵詞:順鉑腎毒性蟬花抗發炎抗氧化
外文關鍵詞:cisplatin nephrotoxicityCordyceps soboliferaanti-inflammationanti-oxidant
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  順鉑是一種 DNA-烷化劑型的有效化療藥物,被廣泛用來治療實質固態癌,例如睪丸、卵巢、子宮及乳癌等。然而,腎毒性是順鉑在治療上限制其劑量使用最主要的副作用因素。近年來許多資料指出發炎反應與氧化傷害參與在順鉑所引起腎毒性的致病機制。過去的研究証實蟲草屬產物具有抗發炎與抗氧化的功用,而蟬花萃取物則可減輕因脂多糖所造成的腎臟損傷,但目前尚無證據顯示蟬花是否對順鉑所引起的腎傷害有保護的作用。因此,在本研究中,以大鼠模式來探討蟬花發酵產物對順鉑誘發腎損傷的保護作用。
  實驗將 SD 大鼠隨機分成控制組、順鉑組、蟬花發酵產物組、順鉑暨蟬花發酵產物低劑量組和順鉑暨高劑量蟬花發酵產物組等五組。順鉑(7.5 mg/kg B.W.)以腹腔注射在犧牲前第四天給予,蟬花發酵產物(50 或 200 mg/kg B.W.)在順鉑注射前 8 天起開始管灌餵食持續 12 天。實驗結果顯示順鉑處理組血清中有較高的尿素氮、肌酸肝和一氧化氮,尿蛋白也顯著升高。同時其腎臟組織有較顯著的丙二醛含量及腎組織誘導型一氧化氮合成酶蛋白質表現顯著提升,而抗氧化酵素(超氧化歧化酶、過氧化氫酶、麩胱苷肽過氧化酶)蛋白質表現則顯著減少。組織學觀察到順鉑造成腎絲球損傷及空泡化。
  在給予蟬花發酵產物的實驗動物對順鉑所造成之腎損傷包括生化指標、酵素活性與蛋白質表現及腎組織異常均有顯著改善。
  綜合以上結果顯示,蟬花發酵產物對順鉑毒性的保護作用可能是透過抗氧化及抗發炎的作用。

Cisplatin is a potent chemotherapeutic DNA-alkylating agent used to treat many types of solid tumors such as testis, ovary, cervix and breast. However, nephrotoxicity is the major side effect representing a dose-limiting factor in cisplatin therapy. Recently, accumulated data pointed out that inflammatory response and oxidative damage involved
in the pathogenesis of cisplatin nephrotoxicity. Previous studies demonstrated that Cordyceps possess the effects of both anti-inflammation and anti-oxidant and the extract of Cordyceps sobolifera(CS) could ameliorate the kidney damage induced by LPS.
However, the protective effect of CS on ciaplatin induced renal injury is not clear. In the present study, we investigated the effect of fermentation product of CS against cisplatin induced renal injury in rats. Male SD
rats were divided into five groups; control group, Cisplatin groups, CS group, Cisplatin + low dose CS and Cisplatin + high dose CS. Cisplatin (7.5 mg/kg B.W.)was injected intraperitoneally 4 days prior to the sacrifice and CS(50 or 200 mg/kg B.W.) was administered once per day for 12 consecutive days by gavage, starting 8 days before cisplatin
injection. The results revealed that significant elevation of serum BUN, creatinine, nitric oxide and urinary protein were observed in animals injected with cisplatin. Cisplatin treated rats also showed a marked increase in renal MDA level and an apparent decrease in the activities of
antioxidant enzymes (SOD, CAT and GPx). The expression of iNOS in kidney was elevated in cisplatin treated animals; on the other hand, the expressions of antioxidant enzymes were obviously declined. The histological examination showed severe glomerular damage. In contrast, supplementation of CS resulted in significant improvements in 4 biochemical markers, enzyme activities, protein expressions and
histological features. The results suggest that CS may attenuate cisplatin induced renal injury through anti-oxidative and anti-inflammatory effects.
目錄
目錄............................................I
圖目錄..........................................V
表目錄...........................................VIII
中文摘要.........................................1
英文摘要.........................................3
第一章 緒論.....................................5
第二章 文獻回顧..................................8
第一節 蟲草屬介紹................................8
(一)、冬蟲夏草(Cordyceps sinensis)............8
(二)、蛹蟲草(Cordyceps militaris).............10
(三)、蟬花蟲草(Cordyceps sobolifera)..........11
(四)、蟲草屬之療效與應用.........................13
第二節 順鉑(Cisplatin)........................16
(一)、順鉑之介紹 ............................16
(二)、順鉑之作用機制及臨床應用....................17
(三)、順鉑與急性腎衰竭...........................19
第三章 研究目的..................................32
第四章 材料與方法................................33
第一節 實驗材料..................................33
第二節 蟬花發酵產物之水萃取物及酒精萃取物之製備及
植物性化學物質含量和抗氧化力分析.............41
第三節 動物實驗設計...............................47
第四節 腎功能指數測定.............................50
第五節 一氧化氮(NO)濃度分析......................51
第六節 TNF-α 之酵素免疫分析.......................52
第七節 組織脂質過氧化分析.........................53
第八節 蛋白質定量分析.............................54
第九節 組織抗氧化酵素活性分析......................55
第十節 尿液蛋白含量測定...........................60
第十一節 西方墨點法分析腎臟組織中誘導型一氧化氮合成酶
(iNOS)、超氧歧化酶(SOD)、過氧化氫酶(CAT)
及麩胱苷肽過氧化酶(GPx)蛋白質表現量.......61
第十二節 組織石蠟切片與化學染色....................64
第十三節 統計分析 .............................66
第五章 結果......................................67
第一節 蟬花發酵產物之水萃取物及酒精萃取物之總多酚和
類黃酮含量及抗氧化力分析....................67
第二節 蟬花發酵產物之投予對大鼠血清中尿素氮(BUN)及
肌酸酐(CRE)的影響........................72
第三節 蟬花發酵產物之投予對大鼠血清中一氧化氮(NO)
生成的影響................................76
第四節 蟬花發酵產物之投予對腎損傷大鼠 TNF-α 之影響..78
第五節 蟬花發酵產物之投予對腎損傷大鼠尿蛋白
(Urine protein)之影響....................80
第六節 蟬花發酵產物之投予對大鼠腎臟組織中脂質過氧化
的影響....................................82
第七節 蟬花發酵產物之投予對大鼠腎臟組織中超氧歧化酶
(SOD)活性的影響...........................84
第八節 蟬花發酵產物之投予對大鼠腎臟組織中過氧化氫
(CAT)活性的影響...........................86
第九節 蟬花發酵產物之投予對大鼠腎臟組織中麩胱苷肽
過氧化酶(GPx)活性的影響...................88
第十節 蟬花發酵產物之投予對大鼠腎臟中誘導型一氧化氮
合成酶(iNOS)、超氧歧化酶(SOD)、過氧化氫酶
(CAT)及麩胱苷肽過氧化酶(GPx)蛋白質表現的
影響......................................90
第十一節 蟬花發酵產物之投予對大鼠組織型態的影響.......94
第六章 討論......................................97
第七章 結論......................................102
第八章 參考文獻...................................103
附表.............................................123
圖目錄
圖一、冬蟲夏草型態..................................9
圖二、蛹蟲草型態....................................10
圖三、蟬花蟲草型態..................................12
圖四、順鉑之結構...................................16
圖五、順鉑作用機制..................................17
圖六、順鉑造成急性腎衰竭的過程.......................20
圖七、順鉑誘發氧化傷害機制圖.........................23
圖八、一氧化氮合成途徑..............................24
圖九、順鉑訊息傳遞途徑..............................26
圖十、抗氧化酵素作用機轉............................27
圖十一、實驗流程設計...............................48
圖十二、蟬花發酵產物之水萃取物及酒精萃取物清除DPPH
自由基能力.................................69
圖十三、蟬花發酵產物之水萃取物及酒精萃取物還原力之比較.70
圖十四、蟬花發酵產物之水萃取物及酒精萃取物螯合亞鐵能力
之比較....................................71
圖十五、不同處理之大鼠血清中尿素氮(BUN)的含量.......73
圖十六、不同處理之大鼠血清中肌酸酐(CRE)的含量.......75
圖十七、不同處理之大鼠血清中一氧化氮(NO)的含量......77
圖十八、不同處理之大鼠血清中腫瘤壞死因子-α(TNF-α)
的含量....................................79
圖十九、不同處理之大鼠尿蛋白含量.....................81
圖二十、不同處理之大鼠腎臟中丙二醛(MDA)的含量........83
圖二十一、不同處理之大鼠腎臟中超氧歧化酶(SOD)的活性..85
圖二十二、不同處理之大鼠腎臟中過氧化氫酶(CAT)的活性..87
圖二十三、不同處理之大鼠腎臟中麩胱苷肽過氧化酶(GPx)
的活性...................................89
圖二十四、不同處理之大鼠腎臟中誘導型一氧化氮合成酶(iNOS)
、超氧歧化酶(SOD)、過氧化氫酶(CAT)及麩胱苷肽
過氧化酶(GPx)蛋白質表現量..................91
圖二十五、不同處理之大鼠腎臟組織病理變化..............95
表目錄
表一、台灣地區十大死亡原因(2011)...................7
表二、脂質過氧化分析實驗配方表.......................54
表三、蟬花萃取物之植物性化學物質成分..................67
附表一、蟬花發酵產物萃取物之抗氧化能力................123
附表二、不同處理之大鼠生化檢測數值....................124
附表三、不同處理大鼠腎臟組織脂質過氧化與抗氧化酵素活性..125
附表四、不同處理大鼠腎臟組織中誘導型一氧化氮合成酶(iNOS)
、超氧歧化酶(SOD)、過氧化氫酶(CAT)及麩胱苷肽
過氧化酶(GPx)蛋白質表現量..................126

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