2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643674摘要：細胞外部刺激會透過G protein-coupled receptor來活化Rho GTPase，進而調控各種細胞的功能，而 guanine nucleotide exchange factors (GEFs)是調控 Rho GTPase 的重要因子之一。GEFs透過結合到G蛋白來啟動Rho相關的訊號傳遞。ARHGEF10是GEFs其中的一員，被認為與神經形態以及神經連結之調節有重要的關係。ARHGEF10藉由將RhoGTPase的GDP轉換為GTP而使Rho蛋白活化。我們是第一個建立了 ARHGEF10基因剔除小鼠，並且與B6小鼠交配了 10代以上，我們從ARHGEF10的基因剔除小鼠初步發現，剔除ARHGEF10基因會降低小鼠對熱刺激及化學發炎刺激引起之疼痛反應，影響血小板的凝血功能以及影響腦中風的病理現象。綜合以上的初步發現，我們將在兩年内完成下列目標：(1)將會利用ARHGEF10基因剔除鼠來探討ARHGEF10在疼痛反應的角色。包括利用腳底熱刺激 儀和閃尾儀來測量小鼠對熱刺激的疼痛耐受度。此外，並利用福馬林測試來研究ARHGEF10 基因易彳除小鼠對於發炎所引起的疼痛反應，並探討c-fos, CGRP和substance P等的蛋白 及mRNA表現探討其機轉。此外，我們也將利用Chung’s model來探討ARHGEF10基因易J除 對神經性病變引起疼痛致敏之影響。(2)將會利用ARHGEF10基因易J除鼠的PRP(platelet rich plasma)來研究在血小板活化到凝 集過程中ARHGEF10扮演的角色。其中包括FeCl3引起的頸動脈栓塞動物模型，以及血小板 在ADP、 thrombin以及thromboxane receptor agonist等刺激下的凝集反應及血小板結 構之改變。(3)將會利用小鼠的缺血性腦中風動物模型來探討ARHGEF10對於腦中風所可能扮演的角色， 並且將會進一步探討ARHGEF10基因剔除小鼠在中風後的神經細胞以及神經膠細胞是否也 有所影響。此外，也會探討ARHGEF10基因剔除對血腦屏障通透性之影響。經由本計晝，我們會更了解ARHGEF10基因對於疼痛反應、心血管疾病及中風所扮演之角色，期 待在這些疾病能開發新的治療策略。<br> Abstract: Rho GTPases play a fundamental role in numerous cellular processes that are initiated by extracellular stimuli that work through G protein-coupled receptors. Rho guanine nucleotide exchange factor (GEF) may form complex with G proteins and stimulate Rho-dependent signals. ARHGEF10 is a member of the GEF family, which are implicated in neural morphogenesis and connectivity and regulate the activity of small Rho GTPases by catalyzing the exchange of bound GDP by GTP. We are the first to generate ARHGEF10 knockout mice and have backcrossed with B6 for more than 10 generations. We have preliminarily found that the thermal- and chemical-induced nociception response and platelet aggregation, thrombus formation and middle cerebral occlusion (MCAO)-induced infarction were greatly reduced in ARHGEF10 knockout mice. In this two years’ project, we will further examine the role and action mechanism of ARHGEF10 involved in the process of nociception, platelet aggregation and pathology of stroke.The following items will be explored,(1)We will use ARHGEF10 knockout mice to examine the role of ARHGEF10 innociception. The plantar test and formalin-induced pain will be evaluated. The mRNA and protein expression of C-fos, CGRP, substance P and opioid peptides in dorsal horn will be measured. Neuropathic pain will also be induced by modified Chung’s model to examine whether neuropathic pain can be affected by ARHGEF10 knockout. (Year 1)(2)We will examine the role of ARHGEF10 in platelet function. FeCb-induced thrombus formation incarotid artery of mice will be evaluated. In addition, platelet aggregation in response to ADP, thrombin, thromboxane receptor agonist and collagen will be measured in platelet-rich plasma derived from wild-type and ARHGEF10 knockout mice. (Year 2)(3)We will examine the role of ARHGEF10 in stroke. Middle cerebral artery (MCA) occlusion modelwill be used to evaluate the protective action mechanism of ARHGEF10 knockout in ischemia-reperfusion-induced neuronal injury. The molecular changes in both neuron and glia will be further investigated. The blood-brain barrier permeability following ischemia/reperfusion will also be evaluated. (Year 2)From this project, we can learn more about the role of ARHGEF10 in pathological pain and stroke. The new therapeutic strategy can thus be developed.