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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 潘建源(Chien-Yuan Pan) | |
dc.contributor.author | Nai-Hsing Yeh | en |
dc.contributor.author | 葉乃馨 | zh_TW |
dc.date.accessioned | 2021-06-15T14:05:15Z | - |
dc.date.available | 2015-08-21 | |
dc.date.copyright | 2015-08-21 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-20 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52048 | - |
dc.description.abstract | 鈣離子在神經細胞訊息傳遞中扮演了非常重要的角色,而這些功能多半是由具鈣離子結合功能的蛋白質進行作用。Neuronal calcium sensor-1 (NCS-1)是表現在神經細胞中且具有細胞膜結合能力的鈣離子感應蛋白,有三個能和鈣離子結合的 EF-hand 結構。之前的研究證實 NCS-1在牛腎上腺嗜鉻細胞 (adrenal chromaffin cells) 與大鼠腎上腺嗜鉻瘤细胞株 (PC12)中會影響胞內囊泡的循環再利用機轉。我們實驗室先前由酵母菌雙雜合實驗中發現,NCS-1與協助神經細胞胞吞作用的Auxilin-1 (Aux-1)具有交互作用,因此觀察此交互作用是否為神經細胞調控胞吞與胞吐作用的關鍵。我們以GST標定NCS-1來進行Pulldown Assay,在不同鈣離子濃度中無法觀察到明確的交互作用,我們推測NCS-1與Aux-1之間的作用可能只發生於特定的實驗條件之下。為瞭解 NCS-1與Aux-1分別對神經傳導的影響,我們將蛋白質表現在初級培養的神經胚胎細胞中,測量神經細胞受刺激後,胞内鈣離子的濃度變化。結果顯示過量表現NCS-1會使目標細胞及下游細胞的反應顯著的下降,NCS-1的變異株皆會促進下游細胞的鈣離子反應,對目標細胞的影響各有不同。表現Aux-1的目標細胞反應下降,並不影響下游細胞的反應。我們推測NCS-1可能在神經細胞的囊泡回收機轉扮演抑制性的角色,NCS-1變異株則是與內生性的NCS-1競爭來影響細胞的反應,而Aux-1雖會抑制神經細胞的鈣離子反應,仍能夠協助囊泡回收機轉。 | zh_TW |
dc.description.abstract | Calcium influx in neurons is an important event in regulating various signaling pathways like neurotransmitter release and neurite outgrowth. Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand Ca2+ binding proteins and expressed mainly in nervous system. NCS-1 overexpression affects the vesicle recycling in bovine adrenal chromaffin cells (BC) and has little effect on Ca2+ channels. In our lab, we have also found that NCS-1 and Auxilin-1 (Aux-1), a protein assists endocytosis, have interaction via yeast-two-hybrid. We believe this interaction can be the key to regulate exocytosis and endocytosis. However, our results in protein pulldown assay shows that NCS-1 and Aux-1 have no direct binding activity. We suppose the interaction only exist under specific condition. To observe the effect of NCS-1 and Aux-1 on neurotransmission, we use Ca2+ imaging technique to visualize Ca2+ response after excitation. Our results demonstrate that NCS-1 overexpression inhibits [Ca2+]i elevation in both presynaptic and postsynaptic neurons, but NCS-1 mutants overexpression can enhance Ca2+ response in postsynaptic neurons and have different effect on target neurons. In the Aux-1 expressing neuron, the [Ca2+]i elevation is reduced, while in postsynaptic neurons is unaltered. Therefore, NCS-1 may play an inhibitory role in neurotransmission by regulating synaptic vesicle recycling, and Aux-1 may suppress Ca2+ response and support synaptic vesicle recycling. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T14:05:15Z (GMT). No. of bitstreams: 1 ntu-104-R02b21010-1.pdf: 1474102 bytes, checksum: f92a794d2b7e3fa8748778702a01b24d (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 致謝 i
摘要 ii Abstract iii 1. Introduction 1 1.1. Synaptic vesicle recycling 1 1.2. Exocytosis 2 1.3. Endocytosis 4 1.3.1. Clathrin-mediated endocytosis 4 1.3.2. Kiss-and-run 6 1.3.3. Activity-dependent bulk endocytosis 6 1.4. Regulation of vesicle recycling and neurodegeneration 7 1.5. Neuron Ca2+ sensor 1 (NCS-1) 7 1.6. Auxilin-1 (Aux-1) 11 2. Material and Method 13 2.1. Cell culture and transfections 13 2.1.1. Primary culture of embryonic cortical neurons 13 2.1.2. PC12 cell 14 2.1.3. E coli. Transformation 14 2.1.4. Plasmid extraction 15 2.1.5. Transfection 15 2.2. Protein pull-down assay 15 2.2.1. Protein expression and purification 15 2.2.2. Pull-down assay 17 2.3. Ca2+ imaging of neuronal cells 18 2.4. Fluorescence imaging with synaptopHluorin 20 2.5. Data analysis 20 3. Results 21 3.1. NCS-1-GST does not interact with Aux-1 21 3.2. NCS-1 does not affect ATP evoked exocytosis in PC12 22 3.3. NCS-1 inhibits neurotransmission in primary cultured cortical neurons 23 3.4. NCS-1 does not affect DHPG evoked neurotransmission in primary cultured cortical neurons 24 4. Discussion 26 4.1. NCS-1 and Aux-1 interaction under yeast-two-hybrid and pull-down assay 26 4.2. NCS-1 regulates iGluR but not mGluR 26 5. Reference 28 6. Figure 32 Scheme 1 Synaptic transmission. 32 Scheme 2 Roles of NCS-1 and Aux-1in exocytosis and endocytosis. 33 Fig. 1 NCS-1 does not interact with Aux-1 in GST pulldown assay. 34 Fig. 2 NCS-1 does not affect ATP evoked exocytosis. 35 Fig. 3 Calcium responses evoked by glutamate in primary cortical neurons. 36 Fig. 4 Normalized Ca2+ response of neurons stimulated by laser-uncaged glutamate. 38 Fig. 5 Normalized Ca2+ response of neurons stimulated by DHPG. 40 | |
dc.language.iso | en | |
dc.title | NCS-1與Auxilin-1在神經細胞中對於訊息傳遞的影響 | zh_TW |
dc.title | Effects of NCS-1 and Auxilin-1 on Neurotransmission in Cultured Cortical Neurons | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王致恬,林崇智 | |
dc.subject.keyword | Auxilin-1,NCS-1,囊泡回收作用,胞吞胞吐作用,初級培養神經細胞, | zh_TW |
dc.subject.keyword | Auxilin-1,NCS-1,synaptic vesicle recycling,exocytosis,endocytosis,primary cultured cortical neurons, | en |
dc.relation.page | 40 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-20 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
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