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研究生:陳俊璋
論文名稱:Cyclosporine 誘導腎損傷引起鎂離子流失與蟬花多醣體之保護作用
指導教授:喬長誠喬長誠引用關係鄭志雄鄭志雄引用關係
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:81
中文關鍵詞:環孢靈蟬花鎂離子
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Cyclosporine (CsA)為著名之免疫抑制劑,廣泛使用於器官移植手
術,以防止人體之排斥作用,目前已有報告指出此藥物易引起肝、腎
功能之損傷,然而,此藥物造成的損傷原因,仍有待探討。
本研究分為兩部分:第一部分探討以 CsA 誘導大鼠的腎臟腎小管
末端的鎂離子通道蛋白損傷程度,停藥之後,觀測腎臟中鎂離子通道
蛋白的自我修復能力。實驗共分為 3 組: (1)正常對照組 (未經過 CsA
處理) ,(2)CsA 組 (每日管餵 CsA 40mg/kg 處理)以及(3)CsA+28 組
(每日管餵 CsA 40mg/kg 處理之後停藥及給與 28 天休息)。結果發現,
CsA 造成腎臟損傷之後,血液中的尿素氮(UN)與肌酸酐(CRE),分別
由正常對照組的 14.40±2.88 與 0.44±0.05mg/dL,顯著的增加至 CsA
的組 24.60±3.05 與 0.52±0.04mg/dL,在給予 28 天修復之後(CsA+28
組),血液中的尿素氮(UN)與肌酸酐(CRE)降低至 11.40±2.19 與
0.42±0.08mg/dL,明顯的有舒緩。然而發現尿素氮清除率(Clearance of
Urea Nitrogen)與肌酸酐廓清率(Creatinine Clearance Rate),在休息 28
天之後,情況有變嚴重的現象。在實驗中並以血液以及尿液中的鎂離
子含量評估腎功能的重吸收能力。在鎂離子排泄量分析中,CsA 組腎
臟中的鎂離子的排泄量明顯的增加,在經過 28 天恢復之後,鎂離子重吸收則有改善現象,唯有腎功能仍未改善。此外,腎臟腎小管末端遠端小管的鎂離子通道蛋白表現方面,TRPM6 和 TRPM7 經過 CsA
誘導之後,表現量顯著降低,經過 28 天休息之後,在腎臟腎小管末
端遠端小管的鎂離子通道蛋白質的表現則有回復情況。
第二部分探討蟬花多醣體對於 CsA 誘導造成腎損傷的鎂離子通
道蛋白之影響。實驗分為 4 組: (1)正常對照組,(2)CsA 組,(3)CSP
組(每日管餵蟬花多醣體 500mg/kg)與(4)CsA+CSP 組(每日管餵 CsA
40mg/kg 與蟬花多醣體 500mg/kg);蟬花保護作用的實驗結果,血液
中的尿素氮(UN),由正常對照組 16.17±3.87mg/dL 顯著的增加至 CsA
組 42.83±33.17mg/dL,然而肌酸酐(CRE)變化不大(0.52±0.04 v.s.
52±0.04 mg/dL),在投予蟬花之後,CsA+CSP 組中 UN 則降低至
27.67±3.67 mg/dL,明顯的具有腎損傷之保護作用,在尿素氮清除率
與肌酸酐廓清率的比較中,腎功能的表現也有回復。此外,在鎂離子
排泄率的部分,CsA 組(5.81± 2.80 mg/dL)經過蟬花多醣體之投予後,
明顯有改善(4.91±2.00 mg/dL),腎臟排泄鎂離子的分率也有降低。
研究結果,證實未給予蟬花多醣體時(只給予 28 天休息),僅能恢
復鎂離子之重吸收,腎功能無法恢復,唯有給予蟬花多醣體後,腎功
能與鎂離子重吸收才能達到改善之效果。在腎臟中的鎂離子通道蛋白
TRPM6 和 TRPM7 經過 CsA 誘導之後,表現量顯著降低,經過蟬花
多醣體投藥或 28 天休息之後,都有恢復的情況。
Cyclosporine (CsA) is one of the most important immune-osuppression drug, which is widely used in organ transplantation to prevent rejection. It has been reported that cyclosporine has renal toxicity and can cause to a
long term adverse effect in renal transplantation. However, the mechanism of renal damages is still need to be explored. The first purpose of this study was to investigate the effect of CsA-induced magnesium selective ion channels (MSIC) damage of renal tubular terminal in kidney and the repair of MSIC after the drug being discontinued on rat. The second purpose is to investigate the effects of Cordyceps sobolifera (CS) extract on the protection of CsA nephrotoxicity with focus on the protection of CsA induced magnesium toxicity.
In the first part of the study, rats were divided into three groups: control group(rats were given distilled water as the control), CsA group (rats were fed CsA 40 mg/kg for 7 days) and CsA recovery group (rats were fed CsA 40 mg/kg for 7 days and continuous 28 days before being sacrified).
Results showed that CsA group significantly increased the levels of BUN and CRE in blood from 14.40±2.88 mg/dL and 0.44±0.05mg/dL to 24.60±3.05mg/dL and 0.52±0.04mg/dL, respectively. After a recovery stage for 28 days, the levels of UN (11.20±2.19mg/dL) and CRE (0.42±0.08mg/dL) in blood were significantly ameliorated. However, a serious phenomenon in both of the levels of clearance of urea nitrogen and creatinine clearance rate was found after a recovery stage for 28 days. In the study of re-abosorption ability of magnesium ion (Mg2+) in renal tubular terminal after the damaging actions of CsA, the excretion levels of
renal functional markers and Mg2+ in urine and blood were significantly in creased, indicating that Cyclosporine induced the waste of magnesium in
CsA treated rats. After 28 days repair, re-absorption of magnesium ions was improved, but renal function were not. Furthermore, the expression of Mg2+ion channel proteins (TRPM6 and TRPM7) in renal tubular terminal was reduced to alower level after the CsA damage. After a 28-day repair,
the proteins expression was shown to be in recovery situation.
In the second part, the study was aimed to assess the protective effect of Cordyceps sobolifera polysaccharides (CSP) on the expression of Mg2+ion channel proteins in CsA-induced renal damage of rats. Rats were divided into four groups: (1) control, (2) CsA (40mg/Kg), (3) CSP (500mg/Kg for 7 days), (4) CsA (40mg/Kg) plus CSP (500mg/Kg for 7 days). Results showed that CsA dramatically increased the levels of UN in blood compared to that of control grpup (16.17±3.87 to 42.83±33.17), whereas the CRE was in a similar level (0.52±0.08 v.s. 0.52±0.04 mg/ dL). In CsA
plus CSP group, the UN level was reduced to 27.67±3.67 mg/dL obtained a significant amelioration result. The clearance of urea nitrogen and creatinine clearance rate were also displayed an obvious recovery. In addition, the excretion rate of Mg2+in CsA group was obviously reduced to a lower level (5.81±2.80 v.s. 4.91±2.00 mg/dL) after the CSP treatment.
This study suggest that the renal function may not be improved if the CSP treatment being absent, only the re-absorption capability of Mg2+ is recovered after a 28-day repair stage. The CsA treatment dose reduce the
expression of Mg2+ion channel proteins (TRPM6 and TRPM7) in renal tissue, while a repair status is obtained distinctly as a Cordyceps sobolifera polysaccharides or result of 28 days recovery stage.
中文摘要........................................................................................................................I
英文摘要......................................................................................................................III
目 錄 ………………………………………………..…………………………………V
圖目錄........................................................................................................................VII
表目錄.......................................................................................................................VIII
第一章 緒論..................................................................................................................1
第二章 文獻回顧..........................................................................................................3
第一節 Cyclosporine...............................................................................3
第二節 Cyclosporine 毒性與鎂離子之關係.......................................5
第三節 蟬花(Cordyceps sobolifera) ....................................................7
第四節 Transient Receptor Potential Melastatin...................................9
第五節 Transient Receptor Potential Melastatin 6.............................10
第六節 Transient Receptor Potential Melastatin 7.............................12
第三章 研究目的.....................................................................................................13
第四章 材料與方法.................................................................................................14
第一節 實驗試劑與材料........................................................................14
第二節 動物實驗設計............................................................................17
第三節 蟬花多醣體萃取流程...............................................................20
第四節 腎功能指數測定........................................................................21
第五節 血液和尿液中鎂離子濃度測定..............................................22
第六節 蛋白質定量分析........................................................................22
第七節 西方墨點法分析腎臟組織中的蛋白質 TRPM6 和蛋白質
TRPM7 含量................................................................................23
第八節 酵素免疫分析法(ELISA) .......................................................24
第九節 免疫組織化學染色(Immunohistochemistry) ........………...24
第十節 總醣含量之測定(phenol-H 2 SO 4 method) ....... …………….25
第五章 實驗結果.....................................................................................................27
第一節 CsA 引起腎損傷後血液與尿液生化值之變化………..…27
第二節 CsA 對於大鼠腎臟排泄尿素氮與肌酸酐分率之比較…..32
第三節 CsA 誘導對大鼠腎臟鎂離子重吸收的影響.......................33
第四節 CsA 對於大鼠腎臟排泄鎂離子排泄分率的影響.............37
第五節 血清中與尿液中的 TRPM6 和 TRPM7 的蛋白質含量....39
第六節 CsA 對於大鼠腎臟之鎂離子通道蛋白 TRPM6 與 TRPM7
之影響..........................................................................................43
第七節 CsA 對於大鼠腎臟之鎂離子通道 TRPM6 與 TRPM7 組織
切片病理影響.............................................................................46
第八節 蟬花多醣萃取物對於大鼠腎臟受 CsA 誘導之後腎功能中
尿素氮與肌酸酐之影響............................................................51
第九節 蟬花多醣萃取物對於大鼠腎臟受 CsA 誘導之後腎臟排泄
肌酸酐廓清率與尿素氮廓清率之影響..................................56
第十節 蟬花多醣萃取物對於大鼠腎臟受 CsA 誘導之後腎臟鎂離
子重吸收的影響.........................................................................59
第十一節 蟬花多醣萃取物對於大鼠腎臟受 CsA 誘導之後腎臟排
泄鎂離子排泄分率的影響.......................................................62
第十二節 蟬花多醣萃取物對於大鼠腎臟受 CsA 誘導之後腎臟之
鎂離子通道蛋白 TRPM6 與 TRPM7 之影響.......................64
第十三節蟬花多醣萃取物對於大鼠腎臟之鎂離子通道 TRPM6 與
TRPM7 組織切片病理影響....................................................66
第六章 結論..............................................................................................................71
第七章 討論..............................................................................................................73
第八章 参考文獻.....................................................................................................78
圖目錄
圖一、Cyclosporin 之結構..........................................................................................7
圖二、蟬花型態............. ..............................................................................................8
圖三、腎臟中鎂離子平衡的調節............................................................................11
圖四、Cyclosporine 腎臟鎂離子流失及自我修復實驗流程..............................17
圖 五 、 蟬 花 對 於 Cyclosporine 腎 臟 鎂 離 子 流 失 及 保 護 作 用 實 驗 流
程.....................................................................................................................18
圖六、尿素氮檢測原理.. ..........................................................................................21
圖七、不同處理之大鼠尿液與血清中尿素氮的含量.......... ............................29
圖八、不同處理之大鼠尿液與血液中肌酸酐的含量...... .................................31
圖九、不同處理之大鼠中尿素氮廓清的過濾量................................................34
圖十、不同處理之大鼠尿液與血清中鎂離子的含量.......................................36
圖十一、 不同處理之大鼠中鎂離子排泄分率(FE-Mg)..................................38
圖十二、 不同處理之大鼠尿液與血清中 TRPM7 蛋白質含量......................41
圖十三、 不同處理之大鼠腎臟中 TRPM7 的排泄分率...... ............................42
圖十四、不同處理之大鼠中的 TRPM6 與 TRPM7 蛋白質含量......................44
圖十五、 不同處理之大鼠中的免疫切片 TRPM6 蛋白質含量.....................47
圖十六、 不同處理之大鼠中的免疫切片 TRPM6 蛋白質含量之量化圖......48
圖十七、 不同處理之大鼠中的免疫切片 TRPM7 蛋白質含量…………….49
圖十八、 不同處理之大鼠中的免疫切片 TRPM7 蛋白質含量之量化圖..... 50
圖十九、不同處理之大鼠尿液中與血清中尿素氮的含量..............................54
圖二十、不同處理之大鼠尿液中與血液中 CRE 的含量.................................55
圖二十一、不同處理大鼠肌酸酐廓清率與尿素氮廓清率檢測含量數值..... 58
圖二十二、不同處理之大鼠尿液與血清中鎂離子的含量.............................. 60
圖二十三、 不同處理之大鼠中鎂離子排泄分率(FE-Mg)............................... 63
圖二十四、不同處理之大鼠中的 TRPM6 與 TRPM7 蛋白質含量..................65
圖二十五、蟬花多醣體處理之大鼠中的免疫切片 TRPM6 蛋白質含量........ 67
圖二十六、蟬花多醣體處理 之大鼠中的免疫切片 TRPM6 蛋白質之量化
圖…………………………………………………………………………….68
圖二十七、蟬花多醣體處理之大鼠中的免疫切片 TRPM7 蛋白質含量........ 69
圖二十八、蟬花多醣體處理之大鼠中的 免疫切片 TRPM7 蛋白質之量化
圖.................................................................................................................... 70
表目錄
表一、蟬花多醣體成分...........................................................................................26
表二、大鼠尿液和血清中的尿素氮與肌酸酐檢測含量數值............................28
表三、不同處理大鼠肌酸酐廓清率與尿素氮廓清率檢測數值.......................32
表四、不同處理之大鼠尿液中與血液中的鎂離子含量....................................34
表五、不同處理之大鼠腎臟中鎂離子排泄的分率...........................................36
表六、大鼠尿液和血清中 TRPM6 與 TRPM7 檢測數值.....................................39
表七、蟬花之大鼠尿液和血清中的尿素氮與肌酸酐檢測含量數值...............52
表八、不同處理大鼠肌酸酐廓清率與尿素氮廓清率檢測含量數值………56
表九、不同處理之大鼠尿液中與血液中的鎂離子含量.. ………………….....59
表十、蟬花之不同處理之大鼠腎臟中鎂離子排泄的分率..............................61
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