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題 名 | 利用細胞培養腎小管模式評估中藥安全性=Evaluation the Chinese Herb Safety in the Cultured Renal Tube |
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作 者 | 陸德齡; | 書刊名 | 中醫藥年報 |
卷 期 | 3 2014.12[民103.12] |
頁 次 | 頁(41)1-(41)44 |
分類號 | 414.5 |
關鍵詞 | 馬兜鈴酸; 辛夷散; 麻黃附子細辛湯; 當歸四逆湯; 厚朴溫中湯三度空間培養腎小管; Honokiol; Aristolochic acid; Shin ti saan extract granules; Ma huang fu tzu his hsin tang extract granules; Dang guei syh nih tong extract granules; Houpo wenzhong tang extract granules; 3D culture tubulogenesis; |
語 文 | 中文(Chinese) |
中文摘要 | 1990-1993年超過 100個婦女服用中藥減肥後引起腎衰竭,研究指出防己中馬兜鈴酸(aristolochic acid)是造成腎毒性的主要成分,在動物模式中,低濃度口服或皮下注射馬兜鈴酸就可導致腎小管上皮細胞壞死,腎臟間質纖維化 (interstitial fibrosis),腎衰竭及尿道癌等不良反應。在馬兜鈴酸事件中另一中藥為厚朴,厚朴對腎毒性仍存有不同之看法,文獻顯示厚朴中成分 honokiol具有抑制腎臟發炎之作用,但也有報告指出 honokiol具細胞毒性,因此造成中藥材有肝腎毒性的疑慮。本計畫欲以三度空間培養腎小管,一個介於動物與單純細胞培養之間的模式評估馬兜鈴酸、含馬兜鈴酸之中藥方劑如辛夷散、麻黃附子細辛湯、當歸四逆湯以及 honokiol、含 honokiol之厚朴溫中湯對腎臟的毒性。將馬兜鈴酸及 honokiol先以平面細胞決定百分之五十致死率藥物濃度,並以此致死率濃度範圍將馬兜鈴酸及 honokiol分成兩種方式加入,一種在培養 MDCK細胞與膠原蛋白時,便混合馬兜鈴酸及 honokiol,視為藥物對腎小管發育之影響;另一種方式待腎小管初步生成後第四日才加入馬兜鈴酸及 honokiol,視為藥物對生成腎小管之影響,於培養第七日以顯微鏡拍照並計數腎小管之數目。結果顯示,以馬兜鈴酸處理三度空間培養腎小管,其抑制腎小管生成之濃度 (約 25-50 µM),相較於抑制平面細胞濃度 (>100 µM),更接近於動物之模式,因此此種三度空間培養模式應可用於測定藥物對腎小管之影響。為測定含馬兜鈴酸中藥方劑如辛夷散、麻黃附子細辛湯、當歸四逆湯科學中藥,是否影響腎臟之毒性,向廠商買來之科學中藥,依照建議量以甲醇萃取並濃縮後計算相對於科學中藥建議量 1倍、 10倍、 100倍劑量,也分成兩種方式加入三度空間培養腎小管,並評估對腎小管生成之影響。結果顯示, 1倍、10倍及 100倍辛夷散、麻黃附子細辛湯、當歸四逆湯、厚朴溫中湯對腎小管生成數目似乎影響不大,但就顯微鏡細部觀察發現,腎小管周圍均有小的細胞碎屑團塊出現,但 100倍劑量投予腎小管小的細胞碎屑團塊出現更嚴重。是否是受損之細胞仍必須進一步由免疫螢光呈色,或經由西方墨點法分析指標蛋白質,以了解是否造成毒性及可能機轉,並更確認藥物之毒性,以確認藥物之安全性。另外,以此模式我們發現厚朴中極低劑量 honokiol測量(約 5-10 µM)對腎小管就有毒性,所以必須進一步確定 honokiol對腎臟之毒性及在植物中含量以確定其安全性。 |
英文摘要 | Over 100 cases of kidney failure in Belgium were attributed to aristolochic acid, the major factor to cause Chinese herb nephropathy, between 1990 and 1993. Low dose of aristolochic acid has been reported to cause epithelia cells of kidney necrosis, interstitial fibrosis, renal failure, and tumors. Honokiol was purified from Magnolia has been reported to arrest cell cycle and cause cell toxicity, on the other hand, honokiol alleviates experimental mesangial proliferative glomerulonephritis. Therefore, honokiol may be considered as a candidate for Chinese herb nephropathy. This proposal used a 3D cultured model of MDCK cells to form renal tubules by which the safety of aristolochic acid, honokiol, and concentrated herbal extracts were evaluated. First, IC50 of aristolochic acid, honokiol and concentrated herbal extracts were determined by 2D cell culture of MDCK cells. Around the IC50 concentrations of aristolochic acid and honokiol were added to the medium containing cells and collagen. After collagen solidification, medium above the collagen containing aristolochic acid and honokiol were replaced every day. After 7 days, count the numbers of tubules and take pictures by microscope to determine the toxicity of drugs on tubule development. To evaluate the toxicity of developed tubules, the drugs were added to tubules after the 4th day from seeding cells into collagen. Our results showed that aristolochic acid inhibits tubulogenesis at the concentration at 25-50 µM. This concentration was lower than the concentration (>100 µM) at 2D culture, indicating that the toxicity of aristolochic acid in 3D cultured model is close to the animal model. Because starch was rich in the concentrated herbal extracts, we extracted the drugs again by menthol. After methanol extraction, the extraction was dried by vacuum and resolved into DMSO. Adding 1X, 10X and 100X suggested doses to cells before and after tubulogenesis, and then counting and taking pictures of the tubules. The results showed that the numbers of tubules after 1X, 10 X and 100X of shin ti saan, ma huang fu tzu his hsin tang, dang guei syh nih tang, dang gui si ni tang, houpo wenzhong tang extract granules treatment were almost the same with control. However, the damage cells were obviously observed around the tubules after 100X extracted drugs treatment. Western blotting and immunostain for cell death markers can be furthered to confirm the damage. In addition, low dose of honokiol caused tubules damage, indicating that honokiol and the Chinese herbs containing honokiol are candidates for tubule safety investigation. |
本系統中英文摘要資訊取自各篇刊載內容。