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中文論文名稱 第一部分:D型絲胺酸之胚胎毒性分析 第二部分:斑馬魚ankh家族在發育時的基因表現
英文論文名稱 PART I : Zebrafish as a model for safety assessments of D-serine PART II:Developmental expressions of two zebrafish ankh genes, ankha and ankhb
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 100
學期 2
出版年 101
研究生中文姓名 陳星光
研究生英文姓名 Xing-Guang Chen
學號 699160247
學位類別 碩士
語文別 中文
口試日期 2012-07-12
論文頁數 80頁
口試委員 指導教授-陳曜鴻
委員-周記源
委員-王芸馨
中文關鍵字 斑馬魚  D型絲胺  鈣離子  NMDA受體  ankha  ankhb  關節炎 
英文關鍵字 Zebrafish  D-serine  calcium  NMDA receptor  ankha  ankhb  arthritis 
學科別分類 學科別自然科學化學
中文摘要 第一部分:D 型絲胺酸之胚胎毒性分析
人體為一有機的生命體,在所有的生命活動過程中,都需要有各種重要的物質元素來參與,而其中鈣離子則廣泛被人探討,因為鈣離子在神經與肌肉的發育上,扮演著關鍵的角色,更為突顯其重要性。目前所知神經突觸末端與肌肉連結的區域會倚賴鈣離子來傳遞訊號,因此,在此區域密集地分布著許多受體,例如像是NMDA受體、AMPA受體、kainate等,以用來幫助鈣離子的傳遞,其中,又以NMDA受體對鈣離子的通透性最高。NMDA受體的活化需要仰賴一些神經傳導物質,而過去研究指出,哺乳動物神經系統中確實存在一種內生性的D型絲胺酸,這種內生性的D型絲胺酸即為活化NMDA受體的關鍵因子之一。在我們的實驗中,透過顯微注射D型絲胺酸到斑馬魚胚胎內,確實會觀察到肌肉纖維與神經分化的不正常,且表現的形態呈現劑量依賴性(dose-dependent),以肌肉方面來說,我們發現高濃度的D型絲胺酸會使魚體外表出現明顯彎曲,且肌肉出現嚴重的排列絮亂,並且在其甩尾運動能力上也大受影響;另外過度表現D型絲胺酸也會使神經分化數量減少,神經排列也比野生種斑馬魚來的不整齊。其表現型態與咖啡因極為類似,更能進一步推測D型絲胺酸與鈣離子釋放的間接關係。因其重要性質,本文將探討D型絲胺酸在神經系統中的分布、合成及其生理機能。而透過顯微注射D型絲胺酸到斑馬魚胚胎之中,觀察D型絲胺酸之胚胎毒性分析。

第二部分:斑馬魚ankh家族在發育時的基因表現
關節病變、僵直性脊椎炎舉凡這一類的關節疾病至今仍是無法明白發生原因,但此疾病卻困擾著許多患者;在脊椎動物方面,研究指出,ANKH基因所表現出來的蛋白為一胺基酸殘基的跨膜蛋白,其表現位置在關節接合處和其他組織,負責細胞內外無機焦磷酸鹽的調控,有助於防止組織鈣化作用,而此同源基因ank在老鼠上的表現也與人類類似,若此基因出現突變,則會導致關節接合處出現結晶。在斑馬魚方面,此基因有兩種表現型態,分別是 ankha與 ankhb,而ankha蛋白是由306個胺基酸所構成,ankhb蛋白則是由501個胺基酸所構成。本研究將透過原位雜交法(whole-mount in situ hybridization)初步探討早期此兩種不同基因在斑馬魚發育時期所表現的位置,實驗結果顯示,ankha與 ankhb在受精後1 cell、6 hpf、12 hpf、18 hpf等早期發育時皆有表現,並在24 hpf、36 hpf、48 hpf、72 hpf、5 dpf、7 dpf訊號主要集中在頭部;不同的是,在24 hpf與36 hpf時,ankhb會表現在卵黃延長區(yolk extension),並且ankhb的表現在5 dpf後訊號便會開始變弱,到7 dpf時幾乎已經無法觀察到訊號表現,這些現象在ankha方面是沒有發現的。
雖然實驗結果無論是ankha或ankhb的表現並不如預期地表現在骨骼相關的位置處,不過也曾有文獻指出,在老鼠方面,早期ank基因也表現在許多軟組織上,這或許與斑馬魚的早期表現相似,將來可再更進一步研究七天後或更大一點時期之斑馬魚的ankh表現模式。
英文摘要 PART I : Zebrafish as a model for safety assessments of D-serine
The area where the nerve were known that synaptic terminals associate with muscle will rely on the calcium ion to transmit the signal, thus, at this area enriched lot of receptor,such as as NMDA receptor, AMPA receptor, kainate, helping the calcium ion transmission.With the permeability among them to calcium ion,the NMDA receptor has the highest permeability.The activation of the NMDA receptor needs to be dependent on some neurotransmitters,studied in the past, the mammal really exist the endogenous D-serine in the nervous system, this endogenous D-serine is one of the key factors for the activation of NMDA receptors In our study, zebrafish embryos by microinjection D-serine is indeed observed abnormal muscle fibers and neural differentiation, and showing the dose-dependent.To muscle,we found high concentrations of D-serine causes the fish appearance significant bending,muscle appearance serious irregular arrangement,and athletic ability is also greatly affected; in addition overexpression of D-serine also cause neuronal differentiation reduce, neural arrangement irregular than the wild species of zebra fish.Expression patterns are extremely similar caffeine,more further speculated that D-serine and calcium release of the indirect relationship. Because the important properties,this study will discuss the D-serine.Utilize through inject D-serine into zebrafish embryo, observe and analyse the D-serine toxicity for zebrafish embryo.

PART II:Developmental expressions of two zebrafish ankh genes, ankha and ankhb
Arthritic is a common condition affecting 3% of people aged 65–69 years,in which calcium containing crystals form in articular cartilage. The crystals most commonly involved consist of either calcium pyrophosphate dihydrate(CPPD) or calcium hydroxyapatite.Although the heritability of the condition in the general community is unknown,it have been described that loss of
function of an inorganic pyrophosphate(PPi) transporter,the ANKH gene, has been demonstrated to
cause excess calcium hydroxyapatite formation in the ank/ank mouse and this defect could be rescued by transfection of the wild-type ank into mutant fibroblasts.Zebrafish has two ANKH homologous genes,respectively ankha and ankhb.In this study whole-mount in situ hybridization wasutilized explore the two different gene signals in zebrafish early developmental stages.
The experimental results show that both of ankha and ankhb are detected in 1 cell, 6 hpf, 12 hpf, 18 hpf of early embryos.In 24 hpf., 36 hpf., 48 hpf., 72 hpf. 5 dpf 7 dpf,the transcript is mainly concentrated in the head;The difference is that at 24 hpf and 36 hpf of ankhb will be manifested in the axial vein,intermediate cell mass and dorsal aorta.5 dpf,ankhb signal is weaker.7 dpf embryo ankha transcript was not detectable in 7 dpf.
論文目次 中文摘要 ………………………………………………………………I
英文摘要 ……………………………………………………………II
目錄……………………………………………………………………V
圖表目錄 ……………………………………………………………IX

第一部分:壹、前言…………………………………………………1
一、鈣在人體中的重要性……………………………………………2
二、人體中的鈣離子…………………………………………………3
三、神經傳導物之鈣離子相關調控與表徵…………………………7
四、神經傳導物質之D型絲胺酸……………………………………8
五、D型絲胺酸之形成……………………………………………10
六、D型絲胺酸之生理功能………………………………………11
七、透過斑馬魚來探討D型絲胺酸的影響………………………12
八、模式物種:斑馬魚……………………………………………12
九、研究動機………………………………………………………13
貳、實驗材料與方法 ………………………………………………14
一、實驗用斑馬魚( Danio rerio )之飼養 ………………………15
二、斑馬魚胚胎收集………………………………………………15
三、顯微注射劑技術………………………………………………16
四、D型絲胺酸條件篩選…………………………………………16
五、甩尾能力記數(in-chorion contraction)…………………………17
六、免疫抗體螢光染色(Whole-mount immunostaining)…………17
七、胚胎包埋及冷凍切片…………………………………………19
八、設備介紹………………………………………………………19
叁、實驗結果…………………………………………………………21
一、D型絲胺酸注射條件與存活率………………………………22
二、注射D型絲胺酸使得斑馬魚胚胎外觀畸形…………………22
三、D型絲胺酸影響慢肌與肌動蛋白的排列……………………24
四、D型絲胺酸影響斑馬魚胚胎的運動能力……………………25
五、D型絲胺酸影響神經…………………………………………26
六、次級運動神經的發育情形……………………………………28
肆、討論………………………………………………………………29
一、D型絲胺酸使鈣離子濃度上升並影響肌肉發育……………30
二、D型絲胺酸影響神經分化……………………………………31
三、D型絲胺酸之未來探討………………………………………32第二部分:壹、前言………………………………………………34
一、結晶沉積性關節病變………………………………………………34
二、ANKH 在脊椎動物上所扮演的角色和功能……………………34
三、ANKH在小鼠之表現情形………………………………………36
四、斑馬魚作為模式物種的優勢………………………………………36
五、斑馬魚之骨骼發育…………………………………………………37
六、研究目的……………………………………………………………37
貳、實驗材料與方法………………………………………………39
一、實驗用斑馬魚(Danio rerio)之飼養………………………………39
二、斑馬魚胚胎收集……………………………………………………39
三、胚胎包埋及冷凍切片……………………………………………39
四、斑馬魚RNA萃取及cDNA合成…………………………………40
五、聚合酶鍊鎖反應(polymerase chain reaction,PCR) ……………41
六、DNA接合反應……………………………………………………41
七、勝任細胞( competent cell )製備與大腸桿菌(Escherichia coli)轉型
…………………………………………………………………………41
八、小量質體萃取……………………………………………………42
九、探針(riboprobe)合成………………………………………………43
十、原位雜交法(whole-mount in situ hybridization)………………43
十一、設備介紹………………………………………………………45
参、結果與討論……………………………………………………46
一、斑馬魚與其他脊椎動物ANKH family之胺基酸序列比較與演化 關係………………………………………………………………46
二、藉由胚胎原位雜交法(whole-mount in situ hybridization)來瞭解斑 馬魚ankh family在時間與空間上的表現………………………47
三、ankha與ankhb表現之異同處……………………………………48
四、斑馬魚與小鼠表現位置之比較…………………………………49
五、脊椎動物與斑馬魚ANKH/ankh family之胺基酸序列與分子結構 具高度保守性……………………………………………………49
六、脊椎動物與斑馬魚ANKH/ankh family表現位置的差異……50
參考文獻………………………………………………………………51
圖表……………………………………………………………………60
Fig.1: 注射D型絲胺酸後之斑馬魚胚胎外觀彎曲角度示意圖……61
Fig.2. 注射D型絲胺酸後27 hpf之斑馬魚胚胎外觀…………………62
Fig.3. 注射D型絲胺酸後48 hpf之斑馬魚胚胎外觀…………………63
Fig.4. 注射D型絲胺酸後之肌肉螢光染色畸形表現……………64
Fig.5. 注射D型絲胺酸後之肌肉螢光染色畸形率統計圖…………65
Fig.6. 注射D型絲胺後之甩尾數統計………………………………66
Fig.7. 運動神經軸突與運動神經肌肉接點染色……………………67
Fig.8. 二級運動神經染色(Zn5)………………………………………68
Fig.9. 神經染色後畸形率統計………………………………………69
Fig.10.D型絲胺酸機制圖………………………………………………70
Fig.11. ankha & ankhb gene演化樹狀圖………………………………71
Fig.12: ankha & ankhb primer位置示意圖……………………………72
Fig.13:不同物種間的ankh胺基酸序列比對…………………………73
Fig.14. ankha & ankhb gene RT PCR…………………………………75
Fig.15. ankha & ankhb mRNA在各時期的表現情形…………………76
Fig.16.ankha & ankhb whole-mount in situ hybridization 切片結果…77
Table.1: D型絲胺酸之不同濃度的存活率與畸形率…………………78
Table.2: ankh gene不同物種間之胺基酸比對………………………79
Table.3: ankha & ankhb gene比較…………………………………80
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