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研究生:陳又任
研究生(外文):You-Ren Chen
論文名稱:小本山葡萄對於人類腎臟近端腎小管細胞之Pgp及MRP2轉運蛋白之影響
論文名稱(外文):Modulation of P-glycoprotein and multidrug resistance-associated protein 2 by Vitis thunbergii in human proximal tubular cells
指導教授:翁芸芳
指導教授(外文):Yune-Fang Ueng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:70
中文關鍵詞:小本山葡萄轉運蛋白藥物交互作用中草藥
外文關鍵詞:PgpMRP2Herb-drug interactionCsACisplatin
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中西藥間產生交互作用是安全用藥的重要條件,藥物運輸排除作用之增強與降低是造成藥物交互作用之重要原因之一。P-醣蛋白 (P-glycoprotein, Pgp) 及multidrug resistance-associated protein 2 (MRP2) 為膜蛋白,是ATP結合運輸蛋白(ATP-binding cassette transporter) 超級家族中的一員,負責許多內生性物質及外來物的運輸。Pgp及MRP2分佈在一些器官中,在腎臟則以近端腎小管的刷狀邊緣膜 (proximal tubular brush border) 為主。
傳統中藥中,小本山葡萄 (Vitis thunbergii) 的根部用於補腎明目、補血活血、舒筋壯骨等作用。有文獻報告中指出,resveratrol所組成的聚合物 (oligomers) 會抑制人類紅血球細胞MRP1運輸蛋白的活性,而小本山葡萄根部也存在著許多resveratrol的聚合物,因此探討小本山葡萄的根部是否會改變腎臟中Pgp或MRP2的活性及表現,而導致藥物交互作用的產生。而以人類近端腎小管HK-2細胞當作實驗材料,利用已知為Pgp的受質Rhodamine 123 (Rh123),來量測小本山葡萄根部萃取物及其活性成分對於Pgp活性的影響。小本山葡萄根部萃取物 (25-100 μg/ml) 及其活性成分 (+)-ε-viniferin (50-100 μM)、ampelopsin C (25-100 μM) 和 (+)-vitisin A (5-100 μM),立即處理HK-2細胞會增加Rh123的累積,在處理72小時,萃取物 (10-15 μg/ml) 及活性成分ampelopsin C (10 μM) 和vitisin A (5-10 μM) 也會增加Rh123的累積。由免疫轉印法 (Immunoblot analysis) 以及reverse transcriptase-polymerase chain reaction (RT-PCR) 的結果可以得知萃取物 (10-15 μg/ml) 及活性成分ampelopsin C (10 μM)、vitisin A (5 -10 μM),處理HK-2細胞72小時,會減少Pgp蛋白質以及mRNA的表現。萃取物及ampelopsin C、vitisin A處理HK-2細胞72小時,並且在48-72小時加入已知為Pgp的受質cyclosporin A (CsA),結果共同處理比單一處理造成較高之細胞毒性。在MRP2方面,以5-carboxyfluorescein (5-CF) 為運輸受質,結果顯示萃取物 (7.5-10 μg/ml) 、viniferin (5-20 μM)、ampelopsin C (2.5-10 μM) 和vitisin A (1-2.5 μM),會在進行efflux的時段中,減少5-CF的排除,而增加處理時間至72小時,萃取物 (10-15 μg/ml) 及活性成分ampelopsin C (10 μM) 和vitisin A (5-10 μM) 也會減少5-CF的排除,萃取物 (10-15 μg/ml) 會減少MRP2 mRNA的表現,但ampelopsin C (10 μM) 和vitisin A (5-10 μM) 卻會增加MRP2 mRNA的表現,其原因並不清楚。萃取物及其活性成分ampelopsin C、vitisin A處理HK-2細胞72小時,並且在48-72小時加入MRP2的受質cisplatin進行測試,結果顯示預處理會增加HK-2細胞對於cisplatin的敏感性。
整體而言,小本山葡萄根部萃取物及其活性成分 ampelopsin C和vitisin A,除了會抑制Pgp的活性,並且會減少Pgp蛋白質以及mRNA的表現。萃取物及其活性成分在立即以及72小時處理也會抑制MRP2的活性。而用已知為Pgp的受質CsA以及MRP2的受質cisplatin當作測試藥物,預處理萃取物及其活性成分的HK-2細胞,會增加HK-2細胞對於CsA以及cisplatin的毒性敏感性,因此在臨床使用時,使用小本山葡萄之病人,共同處理CsA或cisplatin時應注意可能產生之副作用。
Herb-drug interaction is an important factor of the safe use of drugs and modulation of drug efflux functions is one of the main factors of herb-drug interactions. P-glycoprotein (Pgp) and multidrug resistance-associated protein 2 (MRP2) are transmembrane proteins and belong to the ATP-binding cassette (ABC) transporter superfamily, involving in the transport of numerous endogenous and exogenous compounds. Pgp and MRP2 are expressed at the apical surface of epithelial cells of several organs, including kidney. In kidney, Pgp and MRP2 are expressed mainly at the brush border of epithelial cells of proximal tubules.
In traditional Chinese medicine, the root of Vitis thunbergii (VtR) has been used for nourishing kidney, improving eyesight, enriching blood and promoting blood flow and strengthening tendons and bones. Previous reports have demonstrated that resveratrol oligomers are potent MRP1 transporter inhibitor and there are many resveratrol oligomers in VtR. Therefore, this report is to elucidate the effects of VtR extract and its resveratrol oligomers on Pgp and MRP2 activities and expressions and characterize the drug interactions with nephrotoxic transporter substrates. Rhodamine 123 (Rh123) was used as a substrate of Pgp. VtR extract (25-100 μg/ml) or its active ingredients (+)-ε-viniferin (50-100 μM), ampelopsin C (25-100 μM) and (+)-vitisin A (5-100 μM) increased Rh123 accumulation in HK-2 cells immediately. When HK-2 cells were cultured for 72 hr in the presence of VtR extract or its active ingredients, VtR extract (10-15 μg/ml), ampelopsin C (10 μM) and vitisin A (5-10 μM) also increased Rh123 accumulation. Immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses showed that Pgp protein and MDR-1 mRNA were reduced in presence of VtR extract (10-15 μg/ml) and its active ingredients ampelopsin C (10 μM) and (+)-vitisin A (5-10 μM) for 72 hr incubation. VtR extract and its active ingredients exposition could enhance the sensitivity to CsA in HK-2 cells. During the efflux period, VtR extract (7.5-10 μg/ml), (+)-ε-viniferin (5-20 μM), ampelopsin C (2.5-10 μM) and (+)-vitisin A (1-2.5 μM) reduced 5-carboxyfluorescein (5-CF) efflux. When HK-2 cells were cultured for 72 hr in the presence of VtR extract or its active ingredients, VtR extract (10-15 μg/ml), ampelopsin C (10 μM) and vitisin A (5-10 μM) reduced 5-CF efflux. VtR extract (10-15 μg/ml) reduced MRP2 mRNA expression. However, ampelopsin C (10 μM) and vitisin A (5-10 μM) increased MRP2 mRNA expression. VtR extract, ampelopsin C and vitisin A enhanced cellular sensitivity to cisplatin.
In summary, VtR extract and its active ingredients, ampelopsin C and vitisin A, decreased Pgp function and reduced Pgp protein and mRNA levels. Pretreatment of VtR extract and its active ingredients enhance the sensitivity to CsA or cisplatin in HK-2 cells. Thus, possible adverse should be noticed when CsA or cisplatin were used in patients concomitantly treated with VtR.
中文摘要 1

英文摘要 3

緒論 5

實驗材料方法 15

壹、實驗材料 15

貳、實驗方法 18

實驗結果 25

壹、小本山葡萄萃取物對於Pgp之影響 25

貳、小本山葡萄活性成分對於Pgp之影響 27

参、 小本山葡萄萃取物對於MRP2之影響 29

肆、小本山葡萄活性成分對於MRP2之影響 30

討論 32

參考文獻 38

圖表 46

圖目錄
Scheme 1. The picture of Vitis thunbergii. 46

Scheme 2. Structures of resveratrol, (+)-ε-viniferin, ampelopsin C and (+)-vitisin A 47

Figure 1. The HPLC chromatograms of (+)-ε-viniferin, ampelopsin C, (+)-vitisin A and Vitis thunbergii extracts 48

Figure 2. Effect of VtR extract on Rh123 accumulation in HK-2 cells 49

Figure 3. Effect of VtR extract on Rh123 accumulation for 72 hr in HK-2 cells 50

Figure 4. Effect of VtR extract on Pgp protein levels in HK-2 cells 51

Figure 5. Time-course of effect of VtR extract on Pgp protein levels in HK-2 cells 52

Figure 6. Effect of VtR extract on MDR1 mRNA levels in HK-2 cells 53

Figure 7. Effect of VtR extract on the cytotoxicity of CsA in HK-2 cells 54

Figure 8. Effects of resveratrol oligomers on Rh123 accumulation in HK-2 cells 55

Figure 9. Effects of resveratrol oligomers on Rh123 accumulation for 72 hr in HK-2 cells. 56

Figure 10. Effects of resveratrol oligomers on Pgp protein levels in HK-2 cells 57

Figure 11. Time-course of effects of resveratrol oligomers on Pgp protein levels in HK-2 cells. 58

Figure 12. Effects of resveratrol oligomers on MDR1 mRNA levels in HK-2 cells. 59

Figure 13. Effects of resveratrol oligomers on the cytotoxicity of CsA in HK-2 cells. 60
Figure 14. Effect of VtR extract on 5-CF efflux in HK-2 cells. 61

Figure 15. Effect of VtR extract on 5-CF efflux for 72 hr in HK-2 cells. 62

Figure 16. Effect of VtR extract on MRP2 mRNA levels in HK-2 cells. 63

Figure 17. Effect of VtR extract on the cytotoxicity of cisplatin in HK-2 cells. 64

Figure 18. Effects of resveratrol oligomers on 5-CF efflux in HK-2 cells. 65

Figure 19. Effects of resveratrol oligomers on 5-CF efflux for 72 hr in HK-2 cells. 66

Figure 20. Effects of resveratrol oligomers on MRP2 mRNA levels in HK-2 cells. 67

Figure 21. Effects of resveratrol oligomers on the cytotoxicity of cisplatin in HK-2 cells. 68
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Drug Discovery Today 12: 664-673
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