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標題: | 利用活體電穿孔技術在下視丘-神經垂體系統中研究 Synaptotagmin 和 Synapsin Ia 對分泌催產素和血管加壓素所扮演的角色 The Study of the Roles of Synaptotagmin and Synapsin Ia in the Oxytocin and Vasopressin Release from Hypothalamic-Neurohypophysial System by In Vivo Electroporation |
作者: | Hsin-I Jen 任心怡 |
指導教授: | 王致恬(Chih-Tien Wang) |
關鍵字: | 突觸結合蛋白,催產素,血管加壓素,視上核,電穿孔,突觸蛋白, Synatotagmin,oxytocin,vasopressin,electroporation,supraoptic nucleus,synapsin, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 催產素和血管加壓素在許多動物行為中扮演了重要的調控角色,其分泌來源以及功能受到廣泛的研究。我們已知視上核為分泌此兩種荷爾蒙的來源之一,其內部神經細胞可利用軸突及樹突,分別將包覆於緻密核心囊內的催產素和血管加壓素分泌至血液以及腦脊髓液中,但是分泌的機制仍未明瞭。為了釐清此機制,我們首先利用活體電穿孔技術將不同囊泡相關蛋白,包括Synaptotagmin I、IV(Syt I & Syt IV) 以及Syanpsin Ia (Syn Ia) 轉染至大鼠視上核內。Syt I、IV皆為鈣離子感測蛋白,可調控緻密核心囊泡的分泌量以及分泌速度;而Syn Ia為一種可磷酸化蛋白,研究指出其具有調控突觸囊泡與機動蛋白束互動的能力,近來更進一步了解Syn Ia也可以影響緻密核心囊泡的分泌。
在本篇研究中,首先藉由免疫螢光染色,我們證明利用活體電穿孔技術可成功將目標蛋白大量表現在視索上核內。接著,為了觀察此兩種蛋白對於囊泡分泌的機制,我們使用電子顯微鏡觀察緻密核心內囊泡的直徑以及囊泡與細胞膜間距離的改變,同時也利用酵素連結免疫吸附試驗,來觀察血液與腦脊髓液內催產素與血管加壓素的含量,在大量表現不同目標蛋白時有何影響。結果顯示:第一、Syn Ia參與了正向調控腦部受損時血管加壓素的分泌。第二、於視上核大量表現Syt IV不影響血液內催產素含量,卻降低了血液中血管加壓素含量,因此催產素與血管加壓素分泌至血液的調控機制並不相同。第三、於視上核大量表現Syt IV,其軸突分泌至血液內的催產素含量並未改變,但其樹突分泌至腦脊液的催產素含量卻明顯增加,由此可知軸突和樹突也是利用不同調控機制分泌催產素。 最後,我們也發現同時大量表現Syt IV與Syn Ia的點突變蛋白,會導致緻密核心囊泡大量聚集、縮短囊泡與細胞膜間距離以及降低催產素和血管加壓素的分泌量。我們的結果顯示,軸突和樹突是利用不同調控機制分泌催產素且,催產素與血管加壓素分泌至血液的調控機制並不相同。Syt IV與Syn Ia兩者相互作用參與了調控視上核催產素與血管加壓素的釋放機制。 Oxytocin (OT) and vasopressin (VP) are hormones involved in the regulation of a wide variety of behaviors. The effects and release origins of these hormones have been well documented; however, release mechanisms are still elusive. One of the release origins of these hormones is the supraoptic nucleus (SON). In the SON neurons, the OT and VP, enclosed in the dense cored vesicles (DCVs), undergo both axonal release and somatodendritic release into blood and cerebrospinal fluid (CSF), respectively. The release mechanisms are still unclear. To resolve these questions, we overexpressed several vesicle proteins in the SON to study the roles underlying OT and VP release. First, we used newly developed in vivo electroporation technique to transfect various vesicle proteins, i.e. Synaptotagmin I , IV (Syt I & Syt IV), and synapsin Ia (Syn Ia), in the SON region of adult male Sprague-Dawley (SD) rats. Syt I and Syt IV are vesicle proteins acting as Ca2+ sensor in regulated exocytosis that has been found to regulate the kinetics of DCV release recently. Syn Ia, a phosphoprotein that controls synaptic vesicle association with actin filaments, has been reported to regulate DCV release. In this study, we first used immunoflourescence staining to confirm that these vesicle proteins can be successfully overexpressed in the SON region by in vivo electroporation. Second, we used electron microscopy (EM) to further study the morphological changes of the vesicles. We analyzed the vesicle size and the vesicular distance to plasma membrane in each transfected group. Third, to determine the physiological roles of these vesicle proteins, we applied the enzyme-linked immunosorbent assay (ELISA) to measure the changes of OT and VP concentration in plasma and cerebrospinal fluid (CSF), which reflect axonal and somatodendritic release, respectively. We found that Syn Ia increased OT and VP release into blood, and also increased OT release into CSF, suggesting that Syn Ia plays as a positive regulator in both axonal and somatodendritic release. In contrast, Syt IV overexpression in the SON region did not change the OT release into blood, but decreased the VP release into blood, indicating that Syt IV plays as a negative regulator in VP axonal release. Thus, the molecular mechanisms underlying OT and VP release from axonal terminals may be different. Moreover, Syt IV overexpression in the SON region increased the OT release into CSF, but rather into blood, suggesting that Syt IV plays as a positive regulator in OT somatodendritic release. Since Syt IV did not change OT axonal release but regulate OT somatodendritic release positively, these results indicated that different molecular mechanisms may be required for axonal release and somatodendritic release. Lastly, overexpressing Syt IV along with Syn Ia mutant (S62A) in the SON region resulted in vesicle cluster formation, and decreased both axonal and somatodendritic release for OT and VP to blood and CSF. These results suggest that the positive role of Syn Ia in axonal and somatodendritic release of these two neuropeptides may be through phosphorylation of the residue at S62. Moreover, Syn Ia may affect neuropeptide axonal and somatodendritic release by interacting with the molecular components in DCVs, such as Syt IV. In conclusion, our study provided some clues for different mechanisms underlying axonal and somatodendritic release for OT and VP. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48116 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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