臺灣博碩士論文加值系統

帕金森氏症是僅次於阿茲海默症第二常見的神經退化性疾病，主要病理特徵為中腦黑質緻密部的多巴胺神經元漸進性退化導致死亡。F-box protein 7 (FBXO7)的基因變異被認為有可能會引起體隱性早發性帕金森氏症(PARK15)。FBXO7可與SKP1、cullin組成ubiquitin E3 ligase complex，來調節細胞內蛋白質代謝。先前我們的研究發現FBXO7一改變胺基酸的Y52C變異，與帕金森氏症的低風險相關。包含Y52C變異點的FBXO7表現於細胞內時，分解速度顯著降低。人類FBXO7啟動子的調控未被探討過，因此我們以PCR增幅-1240、-694、-538、-438、-202及-56至+261的啟動子片段，置於GFP報告者前，再轉染至HEK-293T細胞表現，藉分析GFP螢光亮度，來探討調控FBXO7表現的cis因子。透過高通量影像及流式細胞儀分析，-694及-202片段的啟動子活性顯著高於-56片段。由於FBXO7表現量的上升對帕金森氏症有保護性，我們建立了-694啟動子片段驅動綠螢光蛋白的Flp-In 293細胞株作為平台，來篩檢可增強FBXO7表現的植物萃取液(工研院提供)。結果發現植物萃取液NTNU-319、379、395、439可顯著提升293及SH-SY5Y細胞中FBXO7表現。進一步檢測發現上述植物萃取液對MPP+引起的細胞毒殺性有保護性，其中植物萃取液NTNU-319、395、439並顯著減緩MPP+引起的粒線體膜電位下降。由於目前帕金森氏症沒有預防或減緩病程的藥物，本研究可能發展出有潛力的治療策略。

Parkinson’s disease (PD), the second most common neurodegenerative disorder, is pathologically characterized by loss of dopaminergic neurons in the substantia nigra of the midbrain. Mutations in the F-box only protein 7 gene (FBXO7), the substrate-specifying subunit of Skp1-Cullin-F-Box (SCF) E3 ubiquitin ligase complex, cause PD-15 (PARK15). Previously we identified an amino acid changed variant Y52C in association with decreased risk of developing PD. Upon expression in cells, Y52C variant displayed significantly reduced rate of decay in cycloheximide chase experiment. The human FBXO7 gene promoter has not been analyzed. To investigate cis elements controlling FBXO7 expression, we cloned FBXO7 promoter fragments from -1240, -694, -538, -438, -202 and -56 to +261 by PCR amplification and placed in front of GFP reporter for transfection assay in HEK-293T cells. The expression of GFP was monitored by both high content analysis and flow cytometry. When the expressed GFP level of the -56~+261 promoter fragment was set as 100%, significantly increased FBXO7 promoter activity was observed with the -202~+261 and -694~+261 fragments by both methods. As the protective role of increased FBXO7 level in PD was implicated, Flp-In 293 cell line expressing GFP reporter driven by FBXO7 -694~+261 promoter fragment was constructed and used as a platform to screen herbal extracts provided by Industrial Technology Research Institute for enhancing FBXO7 expression. Herbal extracts NTNU-319, 379, 395 and 439 were found to increase endogenous FBXO7 protein expression in both Flp-In 293 and SH-SY5Y cells. Treatment of the above herbal extracts protected SH-SY5Y cells against MPP+-induced cell death. In addition, herbal extracts NTNU-319, 395 and 439 significantly alleviated MPP+-induced loss of mitochondrial membrane potential. As lack of treatment to prevent or slow PD progression, the proposed study may provide new insights into the therapeutic approach to PD.

中文摘要 I
Abstract II
壹、 緒論 - 1 -
一、帕金森氏症 - 1 -
(一) 神經病理學 - 2 -
(二)致病原因 - 2 -
(三)致病途徑 - 3 -
(四)治療藥物 - 4 -
二、FBXO7基因 - 5 -
(一) FBXO7基因變異與帕金森氏症 - 5 -
(二) FBXO7的構造與功能 - 6 -
貳、研究目的 - 9 -
參、研究材料與方法 - 10 -
(一) FBXO7啟動子增幅及定序 - 10 -
(二) pEGFP-N1-FBXO7重組質體的建構 - 10 -
(三)細胞培養 - 11 -
(四)轉染(transfection) - 11 -
(五)高通量分析系統 - 12 -
(六)流式細胞儀 - 12 -
(七) pcDNA5/FRT/TO質體建構 - 13 -
(八) Flp-In 293細胞株繼代培養 - 13 -
(九)建立Flp-In 293-FBXO7-EGFP穩定細胞株 - 14 -
(十)植物萃取液篩選 - 14 -
(十一)蛋白質萃取與西方轉漬分析 - 15 -
(十二) MTT assay測量MPP+半抑制濃度(IC50) - 16 -
(十三) MTT assay檢測植物萃取液 - 17 -
(十四)粒線體膜電位測定 - 17 -
(十五)統計分析 - 18 -
(十六)轉錄因子預測 - 18 -
肆、結果 - 19 -
一、確認pEGFP-N1-FBXO7重組質體 - 19 -
二、檢測不同FBXO7啟動子片段驅動EGFP表現的情形 - 20 -
三、建立Flp-In 293 FBXO7-694~+261-EGFP細胞 - 20 -
四、植物萃取液篩檢 - 21 -
五、植物萃取液對內生性FBXO7表現量的影響 - 21 -
六、MPP+誘導的帕金森氏症模式的植物萃取液篩選 - 22 -
七、植物萃取液處理後可減緩MPP+引起的粒線體膜電位下降 - 23 -
伍、討論 - 25 -
陸、參考文獻 - 30 -
柒、附錄圖表 - 38 -

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