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研究生:高梅婷
研究生(外文):Mei-TingKao
論文名稱:以視電圖篩選在果蠅視覺膠細胞中潛在影響視覺傳導之基因
論文名稱(外文):Screening visual transduction related gene in Drosophila glia with electroretinogram
指導教授:邱慈暉
指導教授(外文):Tsyr-Huei Chiou
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:45
中文關鍵詞:視電圖膠細胞視覺傳導核酸干擾神經傳導物質
外文關鍵詞:electroretinogram (ERG)glial cellphototransductionRNAineurotransmitter
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膠細胞 (glial cells) 在神經發育和神經系統功能中扮演重要的角色,包括提供神經細胞養分支持和調節、回收神經傳導物質。前人研究指出,果蠅視覺系統中的膠細胞缺陷會導致感光細胞衰亡。此外,膠細胞也具有調節神經傳導的功能,膠細胞失能,會影響神經傳導物質的接收和釋放,使得神經訊號傳遞失敗。本研究的目的是藉由果蠅視電圖 (electroretinogram, ERG) 作為指標,尋找尚未發表且潛在影響果蠅視覺傳導的膠細胞基因。果蠅具有完善的核酸干擾 (RNA interference, RNAi) 基因片段資料庫,且大多數的基因皆可被獨立操控。我們用repoGal4, tubGal80ts使膠細胞中特定基因於成體時期表現量降低。從335個核酸干擾基因片段中,發現112個基因片段的表現量與不正常的視電圖有關。其中我們再挑選出三個基因為anti-silencing factor 1 (asf1),RNA-binding protein (rbp9),和 disc proliferation abnormal (dpa),分別探討各核酸干擾基因片段的表現狀況。表現asf1核酸干擾基因片段的果蠅,約30%的個體表現出不正常的視電圖,包括開啟和關閉電位缺失 (loss of on/off-transient)、開啟和關閉電位弱化 (weaker on/off-transient)、關閉電位反轉 (reversed off-transient)、或相對較慢恢復到靜止膜電位。表現rbp9核酸干擾基因片段者,只受到些微的影響,僅有一個體表現出關閉電位反轉。表現dpa核酸干擾片段者,隨著成體年齡增加,視電圖會記錄到細胞內隨機釋放的雜訊,此外有四隻個體也表現出不正常的視電圖,分別為二隻個體表現關閉電位弱化和二隻個體相對較慢恢復到靜止膜電位。本研究的視電圖亦常類似於視覺傳導中的神經傳導物質失衡的表現。因此我們認為這三個基因可能表現於突觸間隙周圍的膠細胞,且該膠細胞是負責調節神經傳導物質平衡。本研究結果可再與其他方法結合,有助於未來更深入探討果蠅視覺傳導的機制。
Glial cells play an important role in the development and function of the nervous system. Not only do they provide trophic support they also remove and recycle neurotransmitters for neurons. When glial cells defected in visual system, it would cause photoreceptor degeneration. Alternatively, glial cells regulate fly vision. If glial cells malfunction, it will influence neurotransmitter metabolism in Drosophila visual pathway. Drosophila has a genome-wide transgenic RNAi library, therefore most of genes can be manipulated individually. To find out how might visual glial cells affect visual processing in adult Drosophila, I used repoGal4, tubGal80ts driver to knockdown genes specifically in glia. Using abnormal electroretinogram (ERG) as an indicator, I found that 112 of 335 RNAi lines showed abnormal ERG. Then, three genes were selected. We found that knockdown the gene anti-silencing factor 1 (asf1), RNA-binding protein (rbp9), and disc proliferation abnormal (dpa) were abnormal ERG. With the knockdown of asf1, approximately 30% of the flies showed abnormality such as loss of transient, weaker on/off-transient, reversed off-transient, or taking more time to resting potential. The effect of knockdown rbp9 on flies was little, in all of the flies only one showed reversed off-transient. The flies with knockdown of dpa increased randomly intrinsic noise in ERG recording in an age-dependent fashion. In addition, two flies showed weaker off-transient and two flies took more time to resting potential. The ERG phenotype of knockdown asf1, rbp9, and dpa are similar to the aberrant ERG found in neurotransmitter dysregulated flies. Consequently, I considered that these three genes may affect ERG through synaptic cleft regulation. These information is crucial for further works to understand the potential impact of these genes on Drosophila visual system.
摘要 I
ABSTRACT II
ACKNOWLEDGMENT III
CONTENTS IV
LIST OF TABLE V
LIST OF FIGURES VI
INTRODUCTION 1
Drosophila optic lobe 1
Glial cell 1
Neurodegeneration 3
Synaptic cleft 3
Electroretinogram (ERG) 4
MATERIALS AND METHODS 7
Fly strains and crosses 7
Electroretinogram (ERG) recording 7
Stimulus 8
Statistic 8
RESULTS 9
The ERG of wild-type flies 9
ERG screening 9
Anti-silencing factor 1 (asf1, CG9383) 10
RNA-binding protein 9 (rbp9, CG3151) 11
Disc proliferation abnormal (dpa), CG1616 12
The retinal response of flies to flickering white light 13
DISCUSSION 14
ERG screening 14
Anti-silencing factor 1 (asf1, CG9383) 14
RNA-binding protein 9 (rbp9, CG3151) 15
Disc proliferation abnormal (dpa, CG1616) 15
ERG traces under the different wavelength stimulus 16
The flicker fusion frequency of flies in dark adaptation 16
The method of ERG and transgenic RNAi in Drosophila 17
CONCLUSION 19
REFERENCE 20
TABLES 26
FIGURES 31


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