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系統識別號 U0026-2307201215573700
論文名稱(中文) 新生鼠投予Dexamethasone加重缺氧-缺血造成之腦傷探討
論文名稱(英文) Neonatal dexamethasone treatment exacerbates hypoxic-ischemic brain damage
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 100
學期 2
出版年 101
研究生(中文) 張淦勛
研究生(英文) Kan-Hsun Chang
學號 s26991013
學位類別 碩士
語文別 中文
論文頁數 67頁
口試委員 指導教授-許桂森
口試委員-黃阿敏
口試委員-楊尚訓
中文關鍵字 新生鼠DEX之投予  缺氧缺血之腦傷  GLT-1 
英文關鍵字 neonatal DEX treatment  hypoxic-ischemic brain damage  GLT-1 
學科別分類
中文摘要 Dexmethasone (DEX) 是一種合成的強效醣皮質類固醇,常用來促進早產兒肺泡內的表面張力素之生成以及肺部發育之成熟,進而降低早產兒慢性肺疾病發生率以及其死亡率。而一般來說,在早產兒身上給予高劑量DEX治療的時間點往往是小孩腦部發育成熟一個重要時期,因此大家開始對早期類固醇治療在新生兒的安全性以及長期發展的影響給予更多的關注。儘管早期的DEX給予對於預防以及治療慢性肺疾病有很大的幫助,但早產兒還是有可能因為多重器官的發育不全而面臨遭受缺氧-缺血的危險進而造成嚴重的腦部傷害。從我們先前的研究結果發現,早期暴露過DEX的新生鼠在出生後第七天7 (postnatal day 7, P7) 的時候其海馬迴腦區域藉由cDNA Microarray的實驗可以發現有一些和神經滋養與神經保護相關的基因有表現量上升的情形;因此在這邊我們想要知道究竟早期DEX的暴露是否會去影響其個體對缺氧-缺血造成腦傷之敏感度,並探討底下可能參與的機制。
我們利用在P1到P3間給予遞減劑量的DEX來模擬在臨床上早產兒的類固醇治療,並在P7進行缺氧-缺血的實驗。我們發現新生鼠早期的DEX暴露會去加重缺氧-缺血所造成的腦傷,而這樣的腦傷和早期DEX暴露後造成的麩氨酸再回收能力之減低有關。除此之外,我們還發現,早期暴露過DEX的新生鼠其大腦皮質區的麩氨酸傳送通道蛋白GLT-1 (glutamate transporter-1) 以及GLAST (glutamate aspartate transporter);不管在mRNA或者蛋白之表現量都有明顯的受到抑制。而我們也發現了離子型麩氨酸受體 (N-methyl-D-asparte receptor, NMDA-R) 的次單元NR2A與NR2B在早期DEX暴露後其表現比例並沒有受到影像。同時我們藉由啟動子冷光分析實驗 (promoter luciferase assay) 發現,DEX藉由影響GLT-1啟動子-956到-306的區域來達到抑制GLT-1轉錄活性的效果。另外,藉由在新生鼠做ceftriaxone的處理我們發現可以有效的去增加GLT-1的表現量,同時,若給予DEX時伴隨給予ceftriaxone則可以減緩原本由早期DEX暴露造成之腦傷。
藉由以上實驗結果,我們認為早期使用DEX治療的方式很可能會在往後遭受到缺氧-缺血的時候使神經興奮性毒性的增加進而使腦傷更為嚴重。
英文摘要 The synthetic glucocorticoid dexamethasone (DEX) is frequently used to prevent or lessen the morbidity of chronic lung disease (CLD) by facilitating surfactant synthesis in pulmonary alveolus in premature infants. Typically, high doses of DEX are administered for several weeks, notably during a period of life that is critical for the development of the infant brain. Therefore, growing concern has arisen for the long-term safety of this therapy on the brain development of the child. Although DEX treatment is a powerful way for the prevention and management of CLD, but preterm infants are still under the risk of encountering hypoxic-ischemic stress. Based on our cDNA microarray data, we have found that DEX administration during neonatal development may alter the expression pattern of several genes associated with neurotrophic and neuroprotective functions. Thus, the objective is to evaluate the impact of neonatal DEX treatment on hypoxic-ischemic brain damage and characterize the possible underlying mechanisms.
Using a schedule of tapering doses of DEX similar to that used in premature infants, we demonstrate that neonatal DEX treatment exacerbates hypoxic-ischemic brain damage in the immature rats through a glucocorticoid receptor-mediated mechanism. The influence of neonatal DEX treatment on hypoxic-ischemic brain damage was correlated with a decrease in glutamate reuptake. Furthermore, neonatal DEX treatment decreased the expression of GLT-1 and GLAST mRNA and protein in the cerebral cortex. The expression level of the NMDA receptor subunits NR2A and NR2B was not significantly altered by neonatal DEX treatment. By using promoter luciferase assay, we identified that the decrease of GLT-1 transcriptional activity after DEX treatment was associated to the GLT-1 promoter region form -956 to -306. Pretreatment with ceftriaxone effectively increased the expression of GLT-1 protein and rescued exacerbated hypoxic-ischemic brain damage by neonatal DEX treatment.
In conclusion, these results suggest that neonatal DEX treatment before an episode of hypoxic-ischemia actually enhances the brain injury through an enhanced glutamate-mediated excitotoxicity.
論文目次 考試合格證明 I
中文摘要 (Abstract in Chinese) II
英文摘要 (Abstract in English) V
致謝 VIII
目錄 X
圖表索引 XIII
縮寫檢索表 (Abbreviations) XV
第一章、緒論 (Introduction) 01
一、 早產兒相關疾病與呼吸系統發育 02
二、 Dexamethasone 04
三、 早產兒缺氧-缺血造成之腦部傷害 06
四、 神經興奮性毒性 06
五、 麩氨酸傳送通道蛋白 07
六、 研究目的 08
第二章、材料與方法 (Material and methods) 09
一、 實驗動物 10
二、 動物投藥模式 10
三、 大腦皮質切片製備 11
四、 誘發缺氧-缺血腦傷之動物模式 11
五、 腦傷評估 12
六、 麩氨酸再攝取攝取試驗 14
七、 西方點墨法 17
八、 麩氨酸傳送通道蛋白GLT-1啟動子之報告者
基因建構工程 24
九、 GLT-1啟動子之活性分析 27
十、 統計分析 28
第三章、實驗結果 (Results) 29
一、 新生鼠早期投予DEX對缺氧-缺血之腦傷造成之影響 30
二、 新生鼠早期投予DEX造成大腦皮質區域神經突觸小體
和神經膠狀小體其麩氨酸再回收能力之改變 31
三、 新生鼠早期投予DEX造成大腦皮質區域麩氨酸傳送
通道蛋白表現量之影響 32
四、 麩氨酸傳送通道蛋白的表現量與新生鼠早期投予DEX
所造成缺氧-缺血腦傷程度差異之相關性 33
五、 新生鼠早期投予DEX對大腦皮質區域對離子型麩氨酸
受體次單元NR2A與NR2B之影響 34
六、 新生鼠早期投予DEX對往後其麩安酸傳送通道蛋白
GLT-1表現之長期性影響 34
七、 DEX對麩氨酸傳送通道蛋白GLT-1之基因調控 35
八、 Ceftriaxone對缺氧-缺血造成之腦傷以及
麩氨酸傳送通道蛋白GLT-1之影響 36
第四章、討論 (Discussion) 38
一、 主要研究發現 39
二、 DEX處理與缺氧-缺血腦傷之關係探討 39
三、 DEX處理對GLT-1的影響 40
四、 GLT-1缺乏與腦傷之間關係探討 41
五、 GLT-1的調控與NF-κB之關係 42
六、 DEX可能調控GLT-1的機制 43
七、 結論 44
第五章、圖表 (Figures) 45
第六章、參考文獻 (References) 60
作者簡介 68
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