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    請使用永久網址來引用或連結此文件: https://tkuir.lib.tku.edu.tw/dspace/handle/987654321/51818


    題名: 第一部分: 利用斑馬魚評估對乙醯基氨氛造成之腎毒性;第二部分: Capsulin 在斑馬魚胚胎原腎發育中扮演的角色
    其他題名: 1. Nephrotoxicity assessments of acetaminophen during zebrafish embryogenesis 2. capsulin plays a role during zebrafish kidney development
    作者: 彭熙竹;Peng, Hsi-chu
    貢獻者: 淡江大學生命科學研究所碩士班
    陳曜鴻;Chen, Yau-hung
    關鍵詞: 斑馬魚;對乙醯胺基苯酚;capsulin;腎臟發育;Zebrafish;acetaminophen;capsulin;kidney developement
    日期: 2010
    上傳時間: 2010-09-23 16:10:30 (UTC+8)
    摘要: 利用腎臟細胞表現綠螢光的基因轉殖斑馬魚Tg(wt1b:GFP)可以活體觀察腎臟發育的優勢,將其做為模式物種,觀察對乙醯氨基苯酚造成腎毒性及抑制Capsulin 基因對於腎臟發育的影響。第一部分:對乙醯氨基苯酚是近年來常用的退熱和止痛藥物。過量使用對乙醯氨基苯酚會造成哺乳類動物的肝腎損傷,但是其毒性對於胚胎發育卻所知甚少。浸泡不同的對乙醯氨基苯酚濃度(2.25,22.5,45mM)、時間(12, 24, 36, 48, 60 小時)和起始點(12, 24, 36, 48,60hpf),將腎臟變異的結果分為三種類型: (1)沒影響;(2)中度缺失:原腎管和原腎腎小管彎曲弧度變異及空洞化,原腎絲球腫大或委縮但在正常位置;(3)
    嚴重缺失:除和中度缺失相似外,原腎絲球還依中線分開。結果發現對乙醯氨基苯酚毒性造成的存活率或腎臟變異率,都受到濃度、浸泡時間及起始點的影響。根據多種腎臟部位抗體染色切片及定量PCR 結果發現,對乙醯氨基苯酚是引發細胞凋亡而造成腎毒性。再利用wt1b 探針對浸泡對乙醯氨基苯酚的胚胎做全胚胎原位雜交,發現會造成wt1b 訊號減少及不在正常位置表現。第二部分:Capsulin 為具有basic helix I-loop-helix II(bHLH)結構之轉錄因子。為了深入研究capsulin 如何參與腎臟的發育,首先進行胚胎原位雜交確定
    capsulin 在斑馬魚各時期其表現部位(20,24,31,36,42,48,54hpf),與wt1b 切片(31,48hpf)對照結果發現其沒有表現在腎絲球。再利用capsulin 抗體染色發現注射capsulin MO 會造成原有心臟、原腎管、頭部背側後方表現訊號消失或變形。接著用腎臟細胞會表現綠螢光的基因轉殖斑馬魚,發現注射capsulin MO 劑量越高造成的腎臟變異率越高及存活率下降越嚴重。腎功能分析結果也印證注capsulin MO 會造成其功能下降。根據多種腎臟部位抗體染色切片結果顯示注射capsulin MO 造成腎臟部位細胞數量減少及排列不緊密。再利用早期原腎中胚層wt1b 及wt1a 探針對野生種及capsulin MO 後斑馬魚做全胚胎原位雜核,抑制Capsulin 會造成兩者訊號減少及不在正常位置表現。
    Part1.Acetaminophen(AAP) is widely used as an analgesic and antipyretic drug for decades. In mammals, over-dosed of AAP leads to liver and kidney damages but the toxic effects of
    AAP is little known during embryogenesis. Here, we used a green fluorescenct kidney line, Tg(wt1b:eGFP), as a model to observe the APAP-induced nephrotoxicity dynamically. We carried out a series of exposure experiments with different
    concentrations (2.25, 22.5, 45mM), durations (12, 24, 36, 48, 60 h) and onsets (12, 24, 36, 48, 60hpf). In constrast, embryos displayed malformed kidney phenotypes which were
    classified as three groups: (1) no defects; (2) morderate defects: curved and cystic pronephric tubule (pt) and the pronephric duct (pd) with a cystic and atrophic glomerus(gl) at normal position; (3) severe defects: similar to morderate defects but gl are separated by midline. We found that AAP-induced nephrotoxicity and survival rate reduce depended on the exposure dose, durations and onsets. Immunostaining sections and the q-PCR data showed that AAP-induced nephrotoxicity is caused by apoptosis. Furthermore, whole-mount in situ hybridization revealed embryos after AAP treatment had reductive wt1b expression domains. Taken together, AAP could induce nephrotoxicity during zebrafish development.Part2.capsulin is a bHLH transcription factors which controlled kidney development in vertebrate. First of all, Whole mount in situ hybridization experiments showed that capsulin was detected at mesoderm core of 20, 24, 31, 36, 42, 48, 54 hpf embryos. Comparing with wt1b, capsulin was not detected at gl. Then we used immunostaining to
    showed that capsulin was reduced at heart, and pd. capaulin MO also induced kidney malformation and dis-functions. ZO-1 and DAPI immunostaining data revealed that kidney was decomposed and cell numbers were down regulated by capsulin MO. Molecular evidence showed that wt1a and wt1b were reduced in capsulin-morphant. Here we concluded that capsulin is required for zebrafish kidney development.
    顯示於類別:[生命科學研究所] 學位論文

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