建立活體電穿孔的技術以研究Synaptotagmin在下視丘─神經垂體系統中所扮演的角色

Abstract

Synaptotagmin (Syt)為一種鈣離子的感應蛋白，並且在胞吐作用中調控融合孔的動態。先前的研究發現一種Syt的異構物─Syt IV，可以促使PC12細胞中鈣離子調節的胞吐作用形式由完全融合轉變為kiss-and-run。最近更利用基因剔除鼠的研究得知，Syt IV對於腦垂體後葉中神經末梢的神經肽(神經傳導物質的一種)釋放具有調控者的角色。然而，在成鼠的中樞神經系統內，並沒有一種活體轉染方式，可以將我們欲探討的蛋白質─Syt I和Syt IV大量表現在活體的神經系統內，以研究其對於神經傳導物質的釋放所扮演的角色。在這份研究中，我們首先建立一個活體電穿孔的技術，將欲研究的Syt 基因轉染至中樞神經系統內的下視丘─神經垂體系統(HNS)，以研究Syt I 和Syt IV 在中樞神經系統內神經傳導物質的釋放所扮演的角色。我們將基因利用電穿孔的方式轉染至下視丘的視上核(SON)，藉由下視丘─神經垂體系統的神經末梢位於腦垂體後葉的優點，待兩天基因表現後，將腦組織與腦垂體分別取出以研究基因的表現情形。首先，藉由免疫螢光染色與的技術，得知轉染後的基因以蛋白質的形式分布在視上核及神經垂體。接著，利用西方點墨法來再次證實腦垂體後葉中確實有欲轉染蛋白質的表現。並結合反轉錄即時聚合酶連鎖反應方式，偵測轉染的基因是否以mRNA的形式表現在腦垂體後葉；實驗結果發現mRNA的表現量並無增加，比對蛋白質的表現結果，推測轉染在視上核的基因可能以蛋白質的形式傳送至位於腦垂體後葉的神經垂體，因此local translation不會發生在神經垂體。最後，為了證實利用電穿孔的轉染方式可以偵測已知蛋白質的功能，我們利用酵素連結免疫吸附法(ELISA)，比較血漿中的催產素與血管加壓素(又稱抗利尿激素)在電穿孔轉染Synapsin Ia mutant(可抑制細胞內囊泡傳送至細胞膜)前後的表現量。根據初步的結果，發現過量表現Synapsin Ia mutant具有降低血漿中的神經肽的趨勢，推測轉染成功的Synapsin Ia mutant具有預測的功能及活性，使得含有神經肽的囊泡無法傳送至細胞膜進行釋放。總結這篇研究的成果及重要性，我們證實利用成鼠活體電穿孔的技術，可成功地轉染並表現基因在下視丘─神經垂體系統，這項新的技術將會開啟一扇探討中樞神經系統分子與細胞機制的研究大門，用以研究我們欲探討的Syt在中樞神經系統內所扮演的角色。

Synaptotagmin (Syt) serves as a calcium sensor to modulate fusion pore dynamics during regulated exocytosis. Previous studies showed that one of the Syt isoforms, Syt IV, promotes the mode of exocytosis from full-fusion to kiss-and-run in PC12 cells. A recent study demonstrated that Syt IV plays a role in the regulation of neuropeptide secretion from posterior pituitary nerve terminals. However, there is no suitable transfection for overexpression of the proteins of interest in adult rat central nervous system (CNS) to further delineate the molecular structure-functional relationship. In this study, I establish an in vivo electroporation technique to transfect syt genes into the hypothalamic-neurohypophysial system (HNS) to investigate the role of Syt I and Syt IV in CNS. Adult male Sprague-Dawley rats were used for stereotaxic surgery and in vivo electroporation. Two days following in vivo electroporation, the rats were sacrificed to verify gene expression in supraoptic nucleus (SON) and posterior pituitary. Immunofluorescence staining (IF) was used to examine the distribution of HA-Syt expression in HNS. The IF data showed that in vivo electroporation did successfully deliver genes into HNS, and the proteins of interest were expressed in both SON and posterior pituitary. Western blot (WB) was used to verify the protein expression levels of transfected HA-Syt in the posterior pituitary. The data showed that overexpressed HA-Syt was targeted to posterior pituitary, where the mRNA level of Syt was not significant increased according to real-time PCR data. It thus suggests that local translation is unlikely to occur in the posterior pituitary nerve terminals. In addition, from our preliminary data, the oxytocin (OT) and vasopressin (VP) levels in plasma were decreased after we overexpressed Synapsin Ia mutant (S62A) (to abolish vesicle recruitment to plasma membrane) into HNS, suggesting that the overexpressed proteins remain their function in HNS after in vivo electroporation. As a result, we demonstrate that in vivo electroporation is a useful gene delivery method in HNS, and this method can be used to further investigate the role of Syt isoforms in the CNS in the future.