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研究生:汪佩瑩
研究生(外文):Wang, Pei-Ying
論文名稱:在活體外以及活體內調控多巴胺乙型水解酶蛋白質的表現及活性
論文名稱(外文):Modultion of Dopamine Beta-hydroxylase Protein Level and Activity in vitro and in vivo
指導教授:黃春霖黃春霖引用關係童吉士
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:98
語文別:中文
論文頁數:82
中文關鍵詞:多巴胺乙型水解酶核醣核酸干擾子
外文關鍵詞:Dopamine beta-hydroxylaseRNA interference
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多巴胺乙型水解酶(dopamine β-hydroxylase, DBH)又名正腎上腺素生成酶是兒茶酚胺神經傳遞物中將多巴胺轉變為正腎上腺素(norepinephrine, NE)的酵素。NE主要分布在中樞神經系統中,主要功能與動物驚醒反應、維持血壓恆定和調控心血管功能有關。在DBH基因缺陷的病患中,因無法生成NE和腎上腺素而造成低血壓、心血管功能異常等症狀;且在憂鬱症患者中發現其腦中血清素和NE分泌量不足,或其受器有去敏感化(desensitization)的現象。越來越多的研究證實了NE在中樞扮演著維持心血管功能和維持情緒的重要角色。
本篇研究論文使用一種近年來新興起的研究技術〝核醣核酸干擾 (RNA interference, RNAi)機制〞,以轉錄後抑制的方式降解(knockdown) PC12細胞中DBH蛋白質的表現並測其活性上的改變。實驗結果發現,六條DBH的RNAi作用序列表現出有40%~90%不同程度的抑制能力,而對於Dbh活性上意即NE的含量,也皆有50~75%的抑制作用。另外以慢病毒型Dbh shRNA感染細胞株後同樣發現有相當明顯地抑制NE含量的效力。
另一方面,以大白鼠顱內腦室注射免疫毒殺蛋白(anti-DBH-saporin)破壞其中樞NE神經細胞,經組織染色和ELISA結果顯示,腦中有表現NE的神經細胞和NE含量皆有明顯降低的情形。我們再以此NE神經損傷的大白鼠進行憂鬱症的行為表現的分析和心血管功能測試。實驗結果發現,此動物模式在評估憂鬱症的行為上,如強迫性游泳測試、蔗糖水偏好實驗和活動力的測試中並無表現出憂鬱狀態下的絕望行為、興致喪失和活動力下降等行為。反而在動向行為測試中的直立行為測試上發現差異,該動物直立次數有明顯地下降(p=0.028),進一步去測量大白鼠心血管功能卻發現,該動物的心跳速率和血壓值皆較一般大白鼠有明顯降低的情形(p<0.05)。總括上述結果得知中樞正腎上腺素神經細胞的損傷所造成大白鼠的垂直站立的活動次數降低現象,很可能是由於無法維持在一定血壓所導致。
在未來我們將在活體上比較注射病毒型Dbh shRNA和免疫毒殺蛋白兩者在免疫化學分析上及活體行為上的差異;這將做為RNAi之後應用在臨床治療疾病上的一項評估。
Dopamine β-hydroxylase (DBH), and enzyme also called norepinephrine synthesase, which converts dopamine into norepinephrine (NE) in our sympathoadrenergic catecholamine system. NE plays an important role in maintaining homeostatic cardiovascular function and the status of vigilance. Patients diagnosed with DBH deficiency, suffered from lots of cardiovascular diseases, such as hypotension and arrhythmia, because of the incapability of producing NE and epinephrine (Epi). Some pathological studies also suggested that the secretion of serotonin and NE in depression patients were less than healthy individuals. There are increasing numbers of studies aimed to investigate the physiological function of NE by modulating DBH activity.
In this thesis, we used a novel transcriptional inhibition method, RNA interference (RNAi), to knockdown Dbh protein expression and to test the differences between groups’ activities. The results showed six of DBH RNAi had different levels of protein inhibitory abilities from 40% to 90%; each of sequences had 50% to 75% of effects to decrease the NE amount, which means DBH activities had decreased. Furthermore, we tested Dbh lentivirus-based shRNA by infection; the results show the mount of cellular norepinephrine were decreased obviously.
On the other hand, we also intraventriclely microinjected immunotoxin, anti-DBH-saporin (DSAP), destroy noradrenergic neuron to decrease the NE synthesis in vivo. With immunohistochemistry and ELISA to examine the phenomena, results showed NE positive cells and NE amounts were decreased significantly. Then, we used the animal model to diagnose depression and test orthostatic hypotension behaviors such as forced swimming test, sucrose intake and locomotion. The results showed they did not show any hopelessness, loss of interest and decrease of activities, which to diagnose the depression. However, the behaviors test showed their vertical activities were decreased significantly (p=0.028). To confirm the data, we measured the cardiovascular indexes such as heart rate and blood pressure, which showed they had lower indexes than normal (p<0.05). To summarize those results, because of the cardiovascular dysfunction, the injure of central noradrenergic neurons may cause vertical activities decreased.
Finally, both Dbh knockdown- and noradrenergic neuron-destroyed- rats will be analyzed by immunohistochemistry and several behavior tests, to compare the difference of them; these will estimate the clinical application of RNAi in the future.
目錄……………………………………………………………………………I
圖目錄 ……………………………………………………………………… II
附錄目錄 …………………………………………………………………… III
中文摘要……………………………………………………………………IV
英文摘要 ………………………………………………………………… VI
中英文縮寫對表…………………………………………………………… VII
第一章、緒言……………………………………………………………… 1
第一節、背景…………………………………………………… 1
第二節、正腎上腺素系統與相關疾病………………………… 3
第三節、多巴胺乙型水解酶…………………………………… 8
第四節、核醣核酸干擾機制…………………………………… 9
第五節、anti-Dbh-saporin藥理機制………………………… 12
第六節、實驗目的……………………………………………… 13
第二章、材料方法………………………………………………………… 15
第三章、實驗結果………………………………………………………… 31
第一節、活體外細胞分子上的實驗結果……………………… 31
第二節、活體內注射免疫毒殺蛋白的實驗結果……………… 36
第四章、討論……………………………………………………………… 39
第五章、結論……………………………………………………………… 49
第六章、參考文獻………………………………………………………… 50
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