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標題: | Scn1a基因缺陷影響微膠細胞的功能與活化 The effect of Scn1a mutation on microglia function and activation |
作者: | 陳恩莉 En-Li Chen |
指導教授: | 劉宏輝 Horng-Huei Liou |
關鍵字: | 微膠細胞,促炎性活化態,抗炎性活化態,吞噬作用,Scn1a,Nav1.1,Dravet症候群, Microglia,pro-inflammation,anti-inflammation,phagocytosis,Scn1a,Nav1.1,Dravet syndrome, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | Dravet症候群是一種難治性癲癇,為嚴重的嬰幼兒肌陣攣性癲癇,具有延遲的精神和運動發育、自閉症與認知行為障礙等異常。癲癇發作通常伴隨著腦部發炎的症狀與微膠細胞的活化,而微膠細胞為中樞神經系統中的免疫細胞,不論是在發育中或是成熟的中樞神經系統,微膠細胞都會與神經有許多的連結與調控作用。過去的研究顯示微膠細胞會表達Scn1a基因編碼的第I型電壓門控鈉通道(Nav1.1)。而在Dravet症候群中,大多數病患都是屬於Scn1a基因突變,但Scn1a基因在微膠細胞中的作用仍是未知的。因此我們使用Scn1aE1099X基因轉殖小鼠建構了Dravet症候群的動物模型,在體外培養微膠細胞的方式探討Scn1a基因在微膠細胞上的角色。我們的結果首先發現在Nav1.1缺乏下,微膠細胞的存活率會顯著降低。在形態上則發現在靜態時,Nav1.1缺陷下的微膠細胞會呈現微活化的中間態形態,而不是正常的休息態分支結構。而在促炎細胞因子的表達上,發現在Scn1a的缺陷中,TNF-α、IL-1β和IL-6的mRNA表達均顯著的增加。同樣地,在Scn1a缺陷中,TNF-α和IL-6在細胞內和釋放的細胞外蛋白表現也都顯著增加。然而,抗炎性細胞因子TGF-β和IL-10以及指標基因Arg1的mRNA表達,在Scn1a缺陷中均顯著的降低。此外,我們也發現在Nav1.1缺陷微膠細胞的吞噬能力會減弱。另一方面,在LPS的刺激活化下,Nav1.1缺陷中微膠細胞的形態卻仍呈現微發炎活化的形態,而不是正常的變形蟲活化結構。此外,在LPS的刺激活化下,促炎細胞因子TNF-α、IL-1β和IL-6的mRNA表達在Scn1a缺陷中均顯著的減少。而在LPS刺激活化下的TNF-α和IL-6蛋白表現,以及在IL-4刺激活化下的抗炎細胞因子TGF-β和IL-10的mRNA表達與標誌物Arg1也都顯著的減少。這些結果顯示Scn1a突變體在靜態時會使微膠細胞傾向於更活化發炎的狀態,然而在給予刺激後卻呈現整體功能異常降低的現象,這些現象便可能造成Dravet症候群中異常的病理態,並且可能為癲癇發作的原因之一。 Dravet syndrome is a refractory seizure characterized by severe infant-onset myoclonic epilepsy, delayed psychomotor development and autism-spectrum behaviors. Patients with seizure attacks often follow by brain inflammation, leading to the activation of microglia cell. Microglia, CNS immune cell, have many communication and regulation with neuron in developing and adult CNS, and were observed to express the type I voltage-gated sodium channel (Nav1.1), which is encoded by the Scn1a gene. Loss-of-function mutation in Scn1a gene is the predominant molecular cause in most Dravet syndrome patients. However, the role of Scn1a gene in Microglia is unknown. Here, we constructed an animal model of Dravet syndrome with Scn1aE1099X knock-in (KI) allele transgenic mice, which exhibited epileptic discharges. Our results first show that Nav1.1 deficiency reduced the cell viability of microglia. At quiescence, the morphology of microglia under Nav1.1 deficiency showed an intermediate morphology, instead of normal branched structure, which showed a smaller cell size. The mRNA expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, were upregulated in Scn1a deficiency. Likewise, the intracellular and released extracellular protein level of TNF-α and IL-6 were upregulated in Scn1a deficiency. However, the mRNA expression of anti-inflammatory cytokines, TGF-β and IL-10, and the marker, Arg1, were downregulated in Scn1a deficiency. Furthermore, phagocytic function of microglia was reduced by deficit Nav1.1. On the other hand, the morphology of microglia under Nav1.1 deficiency still showed an intermediate morphology, instead of normal amoeboid activated structure which still showed a smaller cell size during LPS activation. Also, the mRNA expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, were downregulated in Scn1a deficiency during LPS activation. The protein level of TNF-α and IL-6 during LPS activation, and the mRNA expression of anti-inflammatory cytokines, TGF-β and IL-10, and the marker, Arg1 during IL-4 activation, were also all decreased. These results suggested that the Scn1a mutant drove microglia to a more inflamed state at quiescence, and attenuates the overall function of microglia after stimulation which led to an irregular pathology in Dravet syndrome and may be a cause of seizures. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77483 |
DOI: | 10.6342/NTU201803587 |
全文授權: | 未授權 |
顯示於系所單位: | 藥理學科所 |
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