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.

目錄
授權書
口試委員審議通過簽名單
謝誌----------------------------------------------I
中文摘要---------------------------------------II
英文摘要----------------------------------------III
目錄--------------------------------------------IV
圖表目錄-----------------------------------------X
第一部分 斑馬魚胚胎浸泡對乙醯氨基苯酚---------1
第一章 前言---------------------------------------1
1.1 對乙醯氨基苯酚的發展事紀-------------------------------1
1.2 對乙醯氨基苯酚止痛解熱調控機制-------------------------3
1.3 對乙醯氨基酚毒性造成人類的臨床反應 --------------------3
1.4 對乙醯氨基苯酚過量使用造成人類的毒性作用---------------4
1.5 對乙醯氨基苯酚在生物體內的代謝及排出途徑---------------5
1.6 對乙醯氨基苯酚毒性對於肝腎影響之機制-------------------5
1.7 對乙醯氨基苯酚的安全用藥劑量---------------------------6
1.8 腎臟在生物體中的重要性---------------------------------7
1.9 斑馬魚胚胎的原腎發育過程-------------------------------8
1.10 斑馬魚胚胎研究毒物測試的優勢--------------------------9
1.11 本論文的研究目的--------------------------------------9
第二章 材料與方法 ----------------------------------------11
2.1 斑馬魚(Danio rerio)及基因轉殖斑馬魚Tg(wt1b:GFP)飼育---11
2.2 斑馬魚胚胎收集----------------------------------------11
2.3 對乙醯氨基苯酚的浸泡方法及劑量------------------------12
2.4 顯微注射對乙醯氨基苯酚溶液----------------------------12
2.5 4 ZO1 抗體染色(ZO1 immunostaining)--------------------13
2.6 Hematoxylin 和Eosin 染色------------------------------13
2.7 α6F 抗體HRP-DAB 呈色合併hematoxylin 染色-------------14
2.8 斑馬魚RNA 萃取及cDNA 合成-----------------------------15
2.9 定量聚合酶鏈鎖反應(real-time quantitative PCR, Q-PCR) 16
2.10 探針(riboprobe)合成----------------------------------16
2.11 原位雜交法(whole-mount in situ hybridization) -------17
2.12 2TUNEL assay-----------------------------------------18
2.13 切片及樣品製備---------------------------------------19
第三章 結果-----------------------------------------------22
3.1 建立對乙醯氨基苯酚劑量浸泡條件------------------------21
3.2 對乙醯氨基苯酚濃度越高，斑馬魚胚胎存活率越低----------21
3.3 浸泡對乙醯氨基苯酚後造成斑馬魚原腎變異標準訂定--------22
3.4 浸泡對乙醯氨基苯酚濃度越高，斑馬魚原腎的形變越嚴重----23
3.5 浸泡對乙醯氨基苯酚時間越長，斑馬魚原腎的形變越嚴重----23
3.6 浸泡對乙醯氨基苯酚起始點越早，斑馬魚原腎缺失越嚴重----24
3.7 對乙醯氨基苯酚毒性造成斑馬魚原腎組織細胞破裂及形變----25
3.8 對乙醯氨基苯酚毒性使斑馬魚腎絲球基因wt1b 表現位置改變-26
3.9 對乙醯氨基苯酚使細胞凋亡基因表現上升、發炎基因表現下降26
3.10 用TUNEL 證明對乙醯氨基苯酚毒性會造成原腎細胞凋亡-----27
3.11 利用顯微注射技術確認浸泡對乙醯氨基苯酚的吸收劑量-----27
第四章 討論-----------------------------------------------28
4.1 對乙醯氨基苯酚毒性劑量在不同生物體的比較--------------28
4.2 對乙醯氨基苯酚毒性對生物體造成的影響------------------28
4.3 對乙醯氨基苯酚造成的腎毒性機制------------------------29
4.4 斑馬魚胚胎為良好的腎毒性篩藥平台----------------------31
4.5 總結--------------------------------------------------31
第二部分 Capsulin 在斑馬魚胚胎原腎發育扮演的角色----------33
第五章 前言-----------------------------------------------33
5.1 Capsulin 在各物種間的表現位置-------------------------33
5.2 Capsulin 在細胞實驗上所扮演的角色---------------------34
5.3 Capsulin 在脊椎動物發育中扮演的角色-------------------34
5.4脊椎動物腎臟發育過程-----------------------------------35
5.5 Capsulin 在哺乳類動物中腎臟發育所扮演的角色-----------36
5.6 Capsulin 在斑馬魚腎臟發育中所扮演的角色---------------38
5.7 實驗目的----------------------------------------------38
第六章 材料與方法-----------------------------------------40
6.1 斑馬魚(Danio rerio)及基因轉殖斑馬魚Tg(wt1b:GFP)飼育---40
6.2 斑馬魚胚胎收集----------------------------------------40
6.3 顯微注射----------------------------------------------40
6.4 ZO1 抗體染色(ZO1 immunostaining) ---------------------40
6.5 Hematoxylin 和Eosin 染色------------------------------40
6.6 α6F 抗體HRP-DAB 呈色合併hematoxylin 染色-------------41
6.7 基因轉殖魚Tg(wt1b：GFP) 原生性綠螢光合併DAPI 染色-----41
6.8 探針(riboprobe)合成-----------------------------------41
6.9 原位雜交法(whole-mount in situ hybridization) --------41
6.10 切片及樣品製備---------------------------------------41
6.11 Yo-Pro 及PI assay -----------------------------------41
6.12 Capsulin 抗體AP 呈色---------------------------------42
6.13 腎功能分析-------------------------------------------42
第七章 結果-----------------------------------------------44
7.1 Capsulin 之時空分佈-----------------------------------44
7.2 Capsulin 訊號沒有表現在斑馬魚胚胎原腎絲球的位置-------44
7.3 Capsulin 蛋白質在斑馬魚胚胎表現位置-------------------45
7.4 注射Capsulin morpholino 造成斑馬魚胚胎原腎發育變異，且劑量越高變異情形越嚴重--------------------------------------45
7.5 注射Capsulin morpholino 造成斑馬魚胚胎腎功能下降------46
7.6 注射Capsulin morpholino 造成斑馬魚胚胎存活率下降------47
7.7 注射Capsulin morpholino 對斑馬魚胚胎原腎細胞組織較無影響47
7.8 注射Capsulin morpholino 會使斑馬魚胚胎原腎絲球及原腎管細胞數目下降，有可能是因為引發細胞自體凋亡造成--------------48
7.9 抑制Capsulin 基因表現會造成wt1a、wt1b 基因表現位置改變48
7.10 利用抑制Capsulin 基因表現在造成斑馬魚原腎缺失時，頭部肌
肉也無法發育，驗證capsulin MO 有效注入--------------------49
第八章 討論-----------------------------------------------50
8.1 脊椎動物capsulin 之表現時期及位置---------------------50
8.2 抑制小鼠之capsulin 表現對器官發育的影響---------------51
8.3 抑制斑馬魚之capsulin 表現對器官發育的影響-------------52
8.4 Capsulin 在斑馬魚腎臟發育中的角色及與其他基因的調控關係53
8.5 總結--------------------------------------------------54
參考文獻--------------------------------------------------56
圖表------------------------------------------------------65
附錄-----------------------------------------------------101
期刊論文-------------------------------------------------107

圖表目錄
圖示一、斑馬魚胚胎原腎浸泡對乙醯氨基苯酚條件圖
圖示二、對乙醯氨基苯酚濃度越高之斑馬魚胚胎存活率越低
圖示三、對乙醯氨基苯酚後造成斑馬魚原腎變異標準訂定
圖示四、對乙醯氨基苯酚濃度越高使斑馬魚原腎的形變越嚴重
圖示五、對乙醯氨基苯酚浸泡時間越長使斑馬魚原腎形變越嚴重
圖示六、對乙醯氨基苯酚浸泡起點越早使斑馬魚原腎缺失越嚴重
圖示七、 對乙醯氨基苯酚毒性會造成腎絲球及腎管細胞變形及產生
排列不規則的情形
圖示八、對乙醯氨基苯酚毒性造成原腎管細胞破裂及空洞化
圖示九、對乙醯氨基苯酚毒性造成神經管及腎絲球細胞不正常
圖示十、對乙醯氨基苯酚毒性使腎絲球基因wt1b 表現位置改變
圖示十一、用TUNEL 證明對乙醯氨基苯酚毒性會造成原腎細胞凋亡
圖示十二、利用顯微注射技術確認浸泡對乙醯氨基苯酚吸收劑量
圖示十三、斑馬魚capsulin 基因在胚胎發育時期的表現
圖示十四、Capsulin 與腎絲球基因Wt1b 訊號表現位置不同
圖示十五、Capsulin 蛋白質在斑馬魚胚胎表現位置
圖示十六、注射capsulin-MO 造成斑馬魚胚胎原腎發育變異，且同一
觀察時間點劑量越高變異情形越嚴重
圖示十七、注射capsulin morpholino 使斑馬魚胚胎腎功能下降
圖示十八、注射capsulin morpholino 使斑馬魚存活率降低
圖示十九、注射capsulin morpholino 對斑馬魚胚胎中胚層細胞組織較無影響
圖示二十、注射capsulin morpholino 對斑馬魚胚胎原腎管細胞組織較無影響
圖示二十一、Capsulin-MO 對斑馬魚胚胎原腎絲球細胞膜型態沒有明
顯影響
圖示二十二、抑制capsulin 基因表現會造成斑馬魚腎絲球及原腎管細胞數目減少
圖示二十三、注射capsulin morpholino 引發細胞自體凋亡反應
圖示二十四、抑制capsulin 基因表現會造成wt1a、wt1b 基因表現位
置改變
圖示二十五、抑制capsulin 基因表現會造成頭部肌肉及腎臟缺失
表一、浸泡不同濃度之對乙醯氨基苯酚斑馬魚胚胎存活率
表二、浸泡不同濃度之對乙醯氨基苯酚斑馬魚胚胎原腎變異率
表三、浸泡不同時間長短之對乙醯氨基苯酚斑馬魚胚胎原腎變異率
表四、浸泡起始點不同之對乙醯氨基苯酚斑馬魚胚胎原腎變異率
表五、檢測浸泡對乙醯氨基苯酚後胚胎中bcl2、cox2、p53 表現程度
表六、引子
表七、注射capsulin-MO 造成各部位蛋白質表現變異率
表八、注射不同濃度capsulin-MO 後在不同時期原腎變異率
表九、注射不同濃度capsulin-MO 後斑馬魚胚胎存活率
表十、引子

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