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    Please use this identifier to cite or link to this item: http://tkuir.lib.tku.edu.tw:8080/dspace/handle/987654321/105257


    Title: 第一部分 : 以斑馬魚模式探討白藜蘆醇和熊果酸對馬兜鈴酸腎病之防護影響;第二部分 : Topiramate對母體卵子形成與子代軟骨發育不良之影響
    Other Titles: Part I. Nephroprotective role of resveratrol and ursolic acid in aristolochic acid-treated zebrafish;part II. Effects of Topiramate on maternal oogenesis and offspring cartilage dysplasia
    Authors: 丁玉如;Ding, Yu-Ju
    Contributors: 淡江大學化學學系博士班
    陳曜鴻;Chen, Yau-Hung
    Keywords: 腎損傷;白藜蘆醇;雄果酸;卵子形成;癲癇藥;軟骨發育;kidney injury;resveratrol;ursolic acid;oogenesis;Topiramate;cartilage dysplasia
    Date: 2015
    Issue Date: 2016-01-22 14:51:40 (UTC+8)
    Abstract: 第一部分:以斑馬魚模式探討白藜蘆醇和熊果酸對馬兜鈴酸腎病之防護影響根據先前研究指出,馬兜鈴酸導致急性腎損傷主要是因為發炎反應所導致,因此選擇白藜蘆醇(Resv)及熊果酸(UA)這兩個具抗發炎功效之成分去進行預防及治療。我們利用3 ppm 馬兜鈴酸引發急性腎損傷,再利用10 ppm Resv 及UA 進行防護實驗,於胚胎發育至48 hpf 觀察其胚胎腎臟損傷比例是否降低。觀察到10 ppm Resv 或UA 腎臟型態與正常對照組型態相似,腎絲球在體軸中線緊靠,且原腎小管也與正常對照組相似達到預防之效果。腎功能檢測的實驗中,發現利用10 ppm 的Resv 或UA 預防後腎絲球過濾率增加22.19 %和25.14%。進一步觀察發現積血的情況明顯的改善且循環系統也較為正常。利用RT-qPCR 分析,顯示預防組發炎基因的表現有顯著減少(TNFα, mpo),也同時減低細胞凋亡情況(bcl2),腎損傷引發之貧血也得到改善(epo, epor)。實驗中設計了腎衰竭指標(TNFα、kim1 或NF-κB)發現其表現量都顯著降低,經過Resv 預防組其表現量分別為0.36,0.28,和3.36 倍;UA 預防組表現量分別為2.77,0.32,和8.10 倍。表示Resv或UA 預防,可顯著降低體內致炎性細胞激素表達減緩發炎反應而達到預防之效果。而體內ROS 的產生也顯著降低,保護腎臟不受發炎介質影響。利用NMR 分析體內代謝產物,發現其穀胺醯胺和許多胺基酸、肌酐酸、膽鹼、乳酸和氧化三甲胺含量近對照組,顯示腎小管再吸收能力的恢復。在這項研究中,我們利用Resv 或UA 預防後,發現能夠抑制炎症反應,恢復腎功能或其他症狀,使腎損傷情況明顯改善。
    第二部分:Topiramate 對母體卵子形成與代軟骨發育不良之影響Topiramate 是一種FDA 批准的抗癲癇藥物,適用於成人及二歲以上兒童。然而,根據數據顯示服用Topiramate 之孕婦會產下唇顎裂之幼兒。目前Topiramate 致畸胎毒性仍不清楚,本研究以母斑馬魚灌餵0.5 mg/g/day 藥物連續7 天,收集子代並觀察藥物對母體及子代的影響。結果顯示,在母體,Topiramate 會抑制卵母細胞的成熟。實驗組周邊核仁期及卵黃生成期細胞佔卵巢總細胞數比對照組來得高,且也可觀察到閉鎖細胞之增加由此可以證明Topiramate 會降低卵母細胞成熟速率。在子代中,可以觀察到早期胚胎有16.3±15.6%細胞外包卵黃(epiboly)、聚合移動以及伸展移動畸形之情況。進一步利用顯微注射藥物進入胚胎,發現注射0.5,1 或2 ng/ml 的Topiramate 畸形率分別為4.9%,25%和63.4%,顯示出不論餵食母體藥物或直接注射藥物至胚胎內都可以觀察到胚胎早期細胞移動不同程度的影響。後期則可以利用Alcian blue 染色觀察軟骨發育,發現角鰓骨(ceratobranchial)缺失、密克爾氏軟骨(Meckel’s cartilage)、角舌軟骨(ceratohyal)及篩板(ethmoid plate)變形等軟骨異常。並統計軟骨發育異常之比例為0-50 %,平均後其畸形比例為23.0 % (n=14)。利用茜素紅(Alizarin red)染色則觀察到角舌骨、密克爾氏骨與脊椎骨較明顯之礦化程度減弱。由於Topiramate 組母魚的產率及子代早期細胞分裂的問題,為了確定其機轉,透過RT-qPCR 觀察母體卵巢、未分化及分化中之卵子內runx2b、smad4、smad1 及smad5 基因的表達,結果顯示出Topiramate 可能藉由這些基因過度表現而造成骨骼發育及卵巢細胞異常。
    Part I. Nephroprotective role of resveratrol and ursolic acid in aristolochic acid-treated zebrafish Aim of this study was to study the nephroprotective effects of resveratrol (Resv) and ursolic acid (UA) in a zebrafish model. Using two transgenic lines, Tg(wt1b:GFP) and Tg(gata1:dsRed) the subtle changes in the kidney and the red blood cells circulation can be easily recorded. Results showed that both Resv and UA treatment can attenuate AA-induced kidney injury and improve the blood circulation. We used glomerular filtration rate assays to evaluate zebrafish''s renal function and found that both Resv and UA treatment can restore renal function (100% for Mock; 56.1±17.3% for AA-treated; 80.2± 11.3% for Resv+AA; and 83.1± 8.1% for UA+AA, n=15). Furthermore, real time RT-PCR experiments show that pre-treatment with either Resv or UA suppresses pro-inflammatory gene expressions. NMR results revealed that tubular amino acid reabsorption can be improved by Resv or UA treated. In conclusion, our findings reveal that AA-induced nephrotoxicities can be attenuated by pre-treatment with either Resv or UA.
    Part II. Effects of Topiramate on maternal oogenesis and offspring cartilage dysplasia Topiramate is used to treat epilepsy in children and adults. In children, it is indicated for the treatment of Lennox-Gastaut syndrome, a disorder that causes seizures and developmental retardation. Recent clinical data showed that administration of Topiramate in pregnant women caused an increased risk of oral clefts on their new born babies. To further understand the adverse effects of Topiramate, we dosed adult female zebrafish with 0.5 mg/g/day for 7 days, and recorded the phenotypic changes of adult females as well as their offspring. In adults, histopathological examination showed that Topiramate treatment reduced oocyte maturation. Meanwhile, around 23.0% (n=14) of offspring derived from Topiramate-treated adults displayed cartilage deformity, such as ceratobranchial missing, ceratohyal, Meckel’s cartilage and ethmoid plate deformation. Alizarin red staining revealed that the mineralization of ceratohyal, Meckel’s cartilage, and vertebrae were downregulated. From the molecular points of view, real-time PCR demonstrated that the expression levels of smad 1/4/5 and runx2b were upregulated in the Topiramate-treated groups at different sites (ovaries, eggs and 1-cell-stage embryos) in comparison with those of mock-treated controls. Finally, whole-mount in situ hybridization on 8 hpf embryos indicated that endogenous runx2b levels were upregulated. Taken together, we concluded that Topiramate might induce maternal smad1/4/5 and runx2b genes overexpression, and then caused offspring skeletal dysplasia.
    Appears in Collections:[化學學系暨研究所] 學位論文

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