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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王致恬(Chih-Tien Wang) | |
dc.contributor.author | Shih-Yuan Liou | en |
dc.contributor.author | 劉士元 | zh_TW |
dc.date.accessioned | 2021-06-15T12:41:40Z | - |
dc.date.available | 2021-08-24 | |
dc.date.copyright | 2016-08-24 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-07-26 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50460 | - |
dc.description.abstract | 催產素(OT)是一種神經傳導物質,調控著許多生理及行為反應。催產素主要是由下視丘-腦下垂體系統中視上核和室旁核的magnocellular neruons (MCNs)所分泌,而負責製造催產素的MCNs,被稱為催產素神經元。在下視丘-腦下垂體系統中,催產素神經元會將軸突末端投射投射到腦下垂體後葉。催產素會進而經由神經元的樹突和軸突分支釋放到腦脊髓液中,或是沿著軸突主幹分泌到腦下垂體後葉並且進入周邊血液系統中。在下視丘-腦下垂體中,神經傳導物質主要是被包覆在兩種囊泡中,分別是large-dense core vesicles (LDCVs)和microvesicles (MVs),兩者皆受到鈣離子調控的胞吐作用所調節。在先前的研究中,我們發現一種synaptic vesicles (SVs)/MVs的蛋白-Synpsin Ia (Syn Ia)會表現在視上核時,藉由其第62個胺基酸位置的磷酸化會增加被包覆在LDCVs中催產素的釋放。因此,為了更進一步釐清Syn Ia在催產素神經元中對於催產素釋放的影響,我們建立了一套能夠在催產素神經元專一表現基因的技術。首先,我們採用催產素啟動子(563bp)和增強子(IGR 182bp)來建構一個能夠同時專一在催產素神經元表現胞吐作用報導蛋白VAMP2-pHVenus以及Syn Ia的DNA。接著,我們將這些DNA轉染到會分泌催產素的下視丘細胞株-GT1-7 cells中,並且利用免疫螢光染色確認DNA能夠成功表現。利用胞吐作用報導蛋白,發現Syn Ia能夠增加囊泡釋放的機率,但此效應會被無法被MAPK磷酸化的Syn Ia (Syn Ia-S62A)所弱化。此外,我們藉由胞吐作用報導蛋白和鈣離子顯像技術確認Syn Ia不是透過囊泡數目的改變或鈣離子流入量去影響到囊泡的釋放。為了更進一步的將此專一表達在催產素神經元的DNA表現於活體中,我們利用活體電穿孔方式將DNA送入視上核的催產素神經元中。之後再利用免疫螢光染色,發現DNA可成功地被表現在催產素神經元中。這些表現胞吐作用報導蛋白的囊泡會經由催產素神經元的軸突被傳送至腦下腺後葉。綜合以上所述,我們建立了一套能夠專一表現在催產素神經元的技術,並且未來可能透過這個技術來了解在催產素神經元中,Syn Ia對於催產素在樹突和軸突釋放的影響。 | zh_TW |
dc.description.abstract | Oxytocin (OT) regulates various physiological functions and behaviors. OT is mainly secreted from the magnocellular neurons (MCNs) of supraoptic nuclei (SON) and paraventricular nuclei in the hypothalamic-neurohypophysial system (HNS). The MCNs are responsible for producing OT and thus named as oxytocinergic neurons (OT neurons). In HNS, OT neurons project their axon terminals to posterior pituitary. OT is then released into cerebrospinal fluid (CSF) from the somatodendrites or axon collaterals. In addition, OT can be secreted to posterior pituitary and further into peripheral plasma from the main axon terminals. In HNS, there two distinct kinds of vesicles, large-dense core vesicles (LDCVs) and microvesicles (MVs), and the release from both types of vesicles are mediated by Ca2+-regulated exocytosis. In the previous study, we found that Synapsin Ia, a SVs/MVs-specific protein, in the SON can increase LDCV-packaged OT secretion by phosphorylation of the amino acid (Ser-62). In this study, to further clarify the effects of Syn Ia on OT secretion can occur in OT neurons exclusively, we established a cell type-specific gene expression technique in OT neurons. First, we used the 563 bp-promoter and the enhancer (IGR 182 bp) to build up a OT neuron-specific exocytosis reporter, VAMP2-pHVenus. In addition, we transfected the constructs into hypothalamic oxytocinergic GT1-7 cells and performed immunostaining to confirm the successful expression. By live-imaging the exocytosis reporter, we found that Syn Ia increased the releasing probability, but this effect was weakened by the MAPK site-phosphodeficient mutant (Syn Ia-S62A). Furthermore, we verified that Syn Ia did not change the vesicle amounts or Ca2+ entry to affect the vesicle release. To further transfect the OT neuron-specific DNA into the OT neurons of SON, we performed in vivo electroporation in adult male rat brain. By conducting immunostaining, we observed that the successful expression of these constructs in the OT neurons. Moreover, the vesicles expressing the exocytosis reporter can be found in the somadendrites and the axons of OT neurons. In conclusion, we set up a OT neuron-specific gene expression technique. In the future, we can detect how Syn Ia may affect OT secretion from the somadendrites and axons of the OT neurons. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T12:41:40Z (GMT). No. of bitstreams: 1 ntu-105-R03B43027-1.pdf: 7850798 bytes, checksum: 2dc9329daff9e1b1ab9a9d231f34a122 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | Contents
口試委員審定書 i 致謝 ii 中文摘要 iv Abstract vi Abbreviations viii Contents xi Chapter I Introduction 1.1 The hypothalamic-neurohypophysial system 1 1.2 Supraoptic nucleus (SON) 2 1.3 Oxytocin 2 1.4 Oxytocin and the MAPK/ERK1/2 signaling pathway 3 1.5 Large dense-core vesicles and synaptic vesicles 4 1.6 Vesicles in the HNS 5 1.7 Mechanisms of neurotransmitter release 6 1.9 The exocytosis reporter (VAMP2-pHVenus) 7 1.8 Synapsin 8 1.9 Phosphorylation and functions of Syn Ia 9 1.10 In vivo electroporation 10 1.11 The hypothalamic cell line – GT1-7 cells 11 1.12 Objectives of the study 12 Chapter II Materials and Methods 2.1 Animals 15 2.2 Plasmid construction and subcloning 15 2.3 Homemade electrodes 18 2.4 Stereotaxic surgery and in vivo electroporation 19 2.5 Collection of plasma and cerebrospinal fluid (CSF) 20 2.6 Heart perfusion and brain fixation 21 2.7 Immunofluorescence chemistry 22 2.8 Cell culture 24 2.9 Transfection 24 2.10 Immunofluorescence staining 25 2.11 Vesicle imaging and data analysis 27 2.12 Ca2+ imaging and data analysis 29 2.13 Stastics 30 Chapter III Results 3.1 Expression of OT in GT1-7 cells 31 3.2 The exocytosis reporter, VAMP2-pHVenus, can be expressed in GT1-7 cells and identified by the anti-GFP antibody 31 3.3 The OT promoter-driven gene expression is high and stable at 80 hr post transfection 32 3.4 The expression of VAMP2-positive vesicles and pHVenus-positive vesicles remains similar in different transfection groups 33 3.5 Syn Ia was mainly colocalized with SV but little colocalized with LDCVs in GT1-7 cells 35 3.6 Subcellular localization of Syn Ia versus OT in GT1-7cells 36 3.7 The ectopic expression of VAMP2-pHVenus may be targeted to both LDCVs and SVs 37 3.8 pHVenus-positive vesicles may also pack OT 38 3.9 The kinetics of exocytosis is increased by Syn Ia in transfected GT1-7 cells 38 3.10 Overexpressing Syn Ia does not change the amount of vesicles 41 3.11 Similar KCl-induced intracellular Ca2+ rises in transfected GT1-7 cells 41 3.12 DiO injection at the one-sided SON affects the projected axons to posterior pituitary 42 3.13 The exocytosis reporter VAMP2-pHVenus can be expressed in the OT neurons of SON by in vivo electroporation 43 3.14 The exocytosis reporter VAMP2-pHVenus can be expressed in the OT nerve terminals at posterior pituitary by in vivo electroporation 44 Chapter IV Discussion 4.1 The role of Syn Ia in regulating vesicle exocytosis in GT1-7 cells 47 4.2 Syn Ia regulates exocytosis via phosphorylation of Ser-62 by MAPK in GT1-7 48 4.3 Other signal pathways regulate the effects of syn Ia on exocytosis 49 4.4 The OT neuron-specific constructs are transfected into the OT neurons of the HNS by in vivo electroporation 49 4.5 The OT promoter-driven VAMP2-pHVenus are successfully expressed in the oxytocinergic GT1-7 cells and the OT neurons of SON 50 4.6 pHVenus-positive vesicles are found in the OT neurons of SON in the HNS 52 4.7 Effects between Syn Ia and OT 52 4.8 Future directions 53 Chapter V Conclusion 55 References 56 List of Figures Figure 1. The hypothalamic-neurohypophysial system. 65 Figure 2. Magnocellular neurons containing oxytocin or vasopressin in the SON and PVN. 66 Figure 3. LDCVs and MVs in the posterior pituitary nerve terminal of HNS. 67 Figure 4. The exocytosis reporter (VAMP2-pHVenus). 68 Figure 5. Molecular interactions and phosphorylation sites of Synapsin Ia. 69 Figure 6. The changed OT levels in CSF and plasma after transfection. 70 Figure 7. OT neuron-specific constructs. 71 Figure 8. Homemade electrodes for in vivo electroporation. 72 Figure 9. Stereotaxic surgery and in vivo electroporation. 73 Figure 10. SON is the target region in HNS for transfection. 75 Figure 11. Oxytocin is expressed in GT1-7 cells (hypothalamic-derived cell lines). 76 Figure 12. The exocytosis reporter, VAMP2-pHVenus, can be expressed in GT1-7 cells. 78 Figure 13. Fluorescence of pHVenus after transfection. 80 Figure 14. Fluorescence of pHVenus can be detected by the GFP channel of a fluorescence microscope. 81 Figure 15. VAMP2-pHVenus can be expressed by the OT promoter-driven constructs in GT1-7 cells. 83 Figure 16. Subcellular localization of Syn Ia in GT1-7 cells. 85 Figure 17. Syn Ia is little colocalized with OT in GT1-7 cells. 87 Figure 18. The OT promoter-driven VAMP2-pHVenus can target to both SVs and LDCVs in transfected GT1-7 cells. 89 Figure 19. The OT promoter-driven VAMP2-pHVenus can partially target to OT-laden vesicles. 91 Figure 20. The kinetics of exocytosis is increased by Syn Ia in transfected GT1-7 cells. 93 Figure 21. Overexpressing Syn Ia or its phosphomutant does not change the expression level of VAMP2-pHVenus in GT1-7 cells. 95 Figure 22. Similar KCl-induced intracellular Ca2+ rise in transfected GT1-7 cells. 97 Figure 23. The anterograde tracing dye DiO was injected into the one-sided SON and can be defected in the posterior pituitary. 99 Figure 24. The OT promoter-driven VAMP2-pHVenus can be expressed in the transfected side of SON after in vivo electroporation. 101 Figure 25. The OT promoter-driven VAMP2-pHVenus can be expressed in the posterior pituitary after in vivo electroporation. 103 Figure 26. The action of Syn Ia in the regulation of exocytosis after Ser-62 phosphorylation in GT1-7 cells. 105 Figure 27. The effects of Syn Ia on central and peripheral release in the SON of HNS can be verified by the pHVenus-positive vesicles in the somadendrites and axon terminals of OT neurons. 106 List of Tables Table 1. The list of primer 107 Table 2. The list of primary and second antibodies for ICC 108 Table 3. The list of primary and second antibodies for IHC 109 Appendix Appendix 1. VAMP2-pHVenus was expressed in the optic chiasm after in vivo electroporation II | |
dc.language.iso | en | |
dc.title | 建立細胞專一性的基因表現技術探討催產素釋放的調控機制 | zh_TW |
dc.title | Establishment of the cell type-specific gene expression technique to study the regulatory mechanism of oxytocin release | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 盧主欽(Juu-Chin Lu),徐立中(Li-Chung Hsu),胡孟君(Meng-Chun Hu),張懿欣(Yih-Hsin Chang) | |
dc.subject.keyword | 催產素,下視丘-腦下垂體系統,synapsin Ia,胞吐作用報導蛋白,鈣離子顯像技術,活體電穿孔,GT1-7 cells, | zh_TW |
dc.subject.keyword | oxytocin,hypothalamic-neurohypophysial system,synapsin Ia,exocytosis reporter,calcium imaging,in vivo electroporation,GT1-7 cells, | en |
dc.relation.page | 114 | |
dc.identifier.doi | 10.6342/NTU201601465 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-07-27 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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