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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張明富 | |
dc.contributor.author | Yi-Ting Lai | en |
dc.contributor.author | 賴怡廷 | zh_TW |
dc.date.accessioned | 2021-06-08T03:31:54Z | - |
dc.date.copyright | 2019-08-27 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-11 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21355 | - |
dc.description.abstract | 腦部神經物質的傳遞對於調控神經系統扮演了重要的角色,若是神經物質的含量失調則可能會導致情緒、記憶、睡眠失調等等問題,因此探討這些神經傳遞物質傳遞的機制對於研究相關疾病來說是重要的。LMBRD1基因編碼了兩種主要蛋白質分別是LMBD1 和NESI。LMBD1蛋白質由540個胺基酸所組成,其中LMBD1在N端比NESI多了73個胺基酸。除此之外,在實驗室先前研究已經利用正子放射斷層掃描分析發現Lmbrd1+/-小鼠以及野生型小鼠在尾靜脈注射18F-DOPA後,Lmbrd1+/-小鼠相較於野生型小鼠腦部紋狀體18F-DOPA的訊號相對提升,這暗示了Lmbrd1基因的踢除可能會影響dopamine在腦中的運輸。本研究的目的是想找出LMBD1是藉由何種機制參與dopamine的運輸。而在腦部多巴胺是一種重要的神經傳遞物質,先前研究已知,多巴胺轉運蛋白 (dopamine transporter, DAT) 及多巴胺自體受體D2 (dopamine autoreceptor D2, D2R) 是兩種主要負責多巴胺運輸的蛋白質,多巴胺自體受體D2會藉由PKA訊息傳遞路徑、PKCβ-ERK訊息傳遞路徑以及和多巴胺轉運蛋白直接交互作用去調控多巴胺轉運蛋白在細胞膜上的表現量。實驗室先前研究已經知道LMBD1透過羧基端 (433-540 a.a.)和多巴胺轉運蛋白有蛋白質-蛋白質之間的交互作用。本研究同樣利用GST-pull down assay觀察到LMBD1羧基端的extracellular domain (433-493 a.a.)和多巴胺轉運蛋白之間有結合,而LMBD1同樣以羧基端(433-493 a.a.)和多巴胺自體受體D2有結合。另外為了探討LMBD1是否影響了多巴胺轉運蛋白和多巴胺自體受體D2的內化,本研究利用共軛焦顯微鏡觀察到當在細胞株中將Lmbrd1 knockdown時會降低多巴胺轉運蛋白的內化,因此多巴胺轉運蛋白在膜上的表現增加。此結果推測先前研究發現Lmbrd1+/-小鼠腦中有18F-DOPA uptake上升的現象可能是由於Lmbrd1 knockdown時會降低多巴胺轉運蛋白的內化所導致。除此之外,先前研究已指出,當訊息傳遞之後或是當dopamine長時間刺激時,AKT Threonine-308上的磷酸化會被去除,並接著發生clathrin-mediated的多巴胺自體受體D2 內化。因此,本研究先在紋狀體細胞STHdhQ7中將Lmbrd1 knockdown後,以西方墨點法觀察到當給予多巴胺處理時確實有細胞內AKT Threonine-308磷酸化下降的情形發生。之後在共軛焦顯微鏡結果觀察到,當在細胞內將Lmbrd1 knockdown和在野生型細胞株給予多巴胺處理時多巴胺自體受體D2有相似的內化上升現象,但同時發現在沒有多巴胺的處理時Lmrbd1 knockdown組別多巴胺自體受體D2在membrane上表現量較野生型細胞株低。從結果推測LMBD1可能是透過未知的方式影響了AKT Threonine-308上的磷酸化。另外,LMBD1可能也調控了D2R 的recycling進而調節其在膜上的表現量。 | zh_TW |
dc.description.abstract | The transport of neurotransmitters in brain plays a key role in the regulation of nervous system. The imbalance level of neurotransmitters in brain can cause problems with mood, memory, addictions, energy, and sleep. Therefore, investigating the mechanism of neurotransmitter transport is important for understanding the neuron diseases. The limb region 1 domain containing 1 (LMBRD1) gene encodes two major proteins which named as LMBD1 and NESI. The LMBD1 protein encodes 540 amino acid residues and it contains additional 73 amino acid residues in the N-terminus of NESI protein. In addition, our previous data showed that the increase of dopamine level was detected in Lmbrd1+/- mouse striatum by 18F-DOPA PET (positron emission tomography) assay. Therefore, we propose whether the LMBD1 involved in the regulation of dopamine transport. The specific aim is to investigate the mechanism of LMBD1 protein involved in dopamine transport. In brain, dopamine functions as an important neurotransmitter. Previous studies showed that dopamine autoreceptor D2 (D2R) and dopamine transporter (DAT) are responsible for the dopamine transport. D2R regulates the surface expression of DAT by PKA pathway, PKCβ-ERK pathway, and by direct protein-protein interactions. In this study, an extracellular domain at the C-terminus from amino acid residues 433 to 493 of LMBD1 can interact with DAT. In addition, the C-terminus of LMBD1 from amino acid residues 433 to 540 is critical for D2R binding. Our previous study showed that LMBD1 serves as a specific adaptor and regulates the clathrin mediated-internalization of insulin receptor. These results suggest that the LMBD1 may be involved in the regulation of the clathrin-mediated D2R and DAT internalization and its downstream signaling pathways. Confocal images showed that DAT failed to internalize when Lmbd1 knockdown in STHdhQ7 cells under dopamine treatment. These results suggest that LMBD1 might regulate the internalization of DAT, resulting in an increase in the uptake of dopamine in Lmbrd1 knockdown cells. Previous studies showed that D2R internalization may accompany with the reduction of AKT at threonine-308 phosphorylation. In this study, the phosphorylation of AKT at threonine-308 was decreased when Lmbrd1 knockdown in STHdhQ7 cells. However, the internalization of D2R also increased in both wildtype and Lmbrd1 knockdown STHdhQ7 cells under dopamine treatment. In addition, in the absence of dopamine treatment, the expression level of D2R was lower on the plasma membrane in Lmbrd1 knockdown cells. These results suggest that LMBD1 might regulate the phosphorylation of AKT threonine-308 by un-identified pathways. In addition, LMBD1 may regulate the expression level of D2R on plasma membrane through the recycling pathway. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:31:54Z (GMT). No. of bitstreams: 1 ntu-108-R06442021-1.pdf: 14227572 bytes, checksum: b65701d72d7941f340be4d631d9ca7bd (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 誌謝…...…..………………………………….……….…...…………..…..…………III
目次………..…………………………………..……………………...…………….. IV 中文摘要…………..………………………………………………………….…....VIII 英文摘要…………..………………………………………...….…………..………...X 縮寫表………….……………………………………………………….…...….......XII 諸論……………………………………………………..……….………………...…..1 (一) LMBRD1基因之相關研究……...……………...…...……...………...……..….1 (1) LMBRD1基因特性……...………...………...……………...…..….........…..1 (2) Lmbrd1基因剔除小鼠之表型………...………...………...…….........……..2 (3) LMBD1蛋白質之功能……...……………...…………...………………......3 (4) LMBD1與內吞作用 (endocytosis) 相關研究……...……………...…...…4 (5) LMBD1與神經分化相關研究……...………...………...…….......………...5 (6) NESI蛋白質之功能……...………...………...……...………...........……....8 (二) 調控多巴胺運送的分子機制……...……..…...………...……...…..……....….9 (1) 多巴胺轉運蛋白 (dopamine transporter) …...………....……..........……..10 (2) 多巴胺自體受體D2 (dopamine autoreceptor D2) …...………......….……..11 研究主題………...……………...…………………………...……………...………..14 材料來源………...………………………………………………………………...…15 一、 藥品………...……….………………...……………………………………..15 二、 酵素………...…………….…………...……………………………………..18 三、 抗體………...…………………………...………………………….………..18 (1) 初級抗體………...……………………………...…….………………...18 (2) 次級抗體………...……………………...…………………….………...19 四、 細胞培養液及試劑………...…………………………………....…………..19 五、 細胞株………...…………………………...………………………..……….19 六、 套組試劑…...………………………...………………………….…………..20 七、 質體…...………………………...……………………..……….……………20 八、 引子…...…………………………...………….........………………………..21 實驗方法………...……………………………………………………....…………...23 一、細胞繼代培養...……………………………… …….……………… ………23 (1) STHdhQ7....…………………………...……….............………... ……..23 (2) HEK293T…………...…………...………...………........…........………23 二、小鼠胚胎細胞因型鑑定分析. …………...………….....………...…………..24 . 三、勝任細胞 (competent cells)的製備….………...…………...………..……….26 四、細菌轉型(transformation)…………...………...………...…....…...………27 五、質體製備…………...………...………...……...………...……............………28 (1) 質體之小量製備 (Mini-preparation of plasmid DNA) ..………...…….28 (2) 質體之中量製備 (Midi-preparation of plasmid DNA) ..………..……..29 六、DNA轉染 (transfection) ..………...……...……… ..………...……. ………30 七、GST融合蛋白質之表現與純化……...…...………… ..………...……. ……..31 八、GST融合蛋白質純化……...…………… ..……....…...…………… ………32 九、Coomassie brilliant blue (CBR)染色法……...…………… ..………… .…....32 十、GST pull-down assay……...…………… ..……......…...…………….. ……..33 十一、西方墨點法 (Western blot analysis) ……...…………… ..………… ……..34 (1) 細胞內蛋白質之分離……...…………… .. …….......…………………...34 (2) 蛋白質定量……...……………………………............….. .……………..35 (3) 正十二烷硫酸鈉-聚丙烯醯銨膠體電泳(SDS-PAGE)………….……..35 (4) 蛋白質轉漬(protein transfer)…………………...……………….……..36 十二、免疫螢光染色 (immunofluorescence staining assay) ……………...….....37 實驗結果………...…………………………………………………………………...39 一. LMBD1與多巴胺轉運蛋白之交互作用關係……………….....………......39 二. LMBD1與多巴胺自體受體D2之交互作用關係………...………………...40 三. 利用shRNA knocked down STHdhQ7細胞株中的LMBD1表現量以探討對自體受體D2內化的影響…………………………………………………41 四. 利用shRNA knocked down STHdhQ7細胞株中的LMBD1表現量以探討對多巴胺轉運蛋白內化的影響…………………………......……………...43 五. 在STHdhQ7細胞株中利用GST-LMBD1(433-493) 以及GST-LMBD1(433-540) 蛋白質競爭內生性LMBD1 以觀察對多巴胺自體受體D2內化的影響…………………………..………………..………………..….……..……44 六. 在STHdhQ7細胞株中利用GST-LMBD1(433-493) 以及GST-LMBD1(433-540) 蛋白質競爭內生性LMBD1 以觀察對多巴胺轉運蛋白內化的影響…………………………………………………......……………………...45 討論…………………………………………………………………..………………47 圖表…………………………………………………………………..……….……...51 附圖…………………………………………………………………..……….……...73 參考文獻………...…………………………………………………………………...76 | |
dc.language.iso | zh-TW | |
dc.title | LMBD1蛋白質調控多巴胺運輸之機制 | zh_TW |
dc.title | The mechanism of LMBD1 protein involved in dopamine transport | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林敬哲,歐展言,陳炳宏 | |
dc.subject.keyword | 多巴胺運輸, | zh_TW |
dc.subject.keyword | dopamine transport, | en |
dc.relation.page | 80 | |
dc.identifier.doi | 10.6342/NTU201903117 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2019-08-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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