臺灣博碩士論文加值系統

老年性黃斑部病變是老年族群中導致視力喪失的重要疾病之一，其成因被認為與視網膜色素上皮細胞內，受到NF-κB (p65) 路徑所調控的發炎性病變有關。目前發現預防老年性黃斑部病變惡化的酵素的表現是有提高視網膜色素上皮細胞抗發炎 / 抗氧化的效果。茄紅素已經被證實對許多人類的退化性疾病，有抗發炎 / 抗氧化的效果，而具有保護作用，但目前不清楚茄紅素對老年性黃斑部病變是否也有保護作用。我們的研究發現，人類視網膜色素上皮細胞 (ARPE-19)，經由TNF-α的誘導，會有NF-κB (p65) 活化，ICAM-1的表現量增加，以及THP-1 (單核球) 黏附現象提高的情形。但在以茄紅素預處理後，可降低NF-κB (p65) 轉位入核及轉錄能力，因而使貼附因子ICAM-1表現下降，單核球黏附現象下降，因此具有抗發炎的效果。另一方面，茄紅素可降低過氧化氫造成的氧化壓力，及其所造成的視網膜色素上皮細胞損傷。其抗氧化作用的機制是來自茄紅素誘導Nrf-2活化，提高ARE 基因的轉錄活性，增加細胞內GCLC mRNA的表現，而增加細胞內的抗氧化酵素glutathione的含量。因此我們結論，在人類視網膜色素上皮細胞，茄紅素具有抗發炎，以及降低氧化壓力所造成的細胞損傷的作用，可能有治療或預防老年性黃斑部病變的效果。

Aged related macular degeneration (AMD) is one of the most common disease which leads to blindness in elderly people. It had been documented that AMD was retinal pigment epithelial (RPE) cells an inflammative disease, which was modulated by NF-κB (p65) pathway in. A lot of the enzyme which could prevent the progression of AMD had antiinflammative / antioxidative effect in RPE cells. Lycopene, which had antiinflammative / antioxidative effect, had been proven to protect human from several degenerative disease. However, it was not clear that if Lycopene could also have protective effect on AMD. Our study found that TNF-α could induce NF-κB (p65) activation, ICAM-1 expression and THP-1(monocyte) adhesion in human retinal pigment epithelial cell (ARPE-19). However, after pretreatment with lycopene, the translocation of NF-κB (p65) into nucleus, the expression of ICAM-1 and the adhesion of THP-1 were all declined. On the other hand, lycopene could reduce the H2O2-induced oxidative stress, and the damage of RPE cell. The antioxidative effect of lycopene came from the induction of Nrf-2 activation, the transcription of ARE gene, the expression of GCLC mRNA, and the expression of glutathione in RPE cell. In conclusion, in the human RPE cell, lycopene had anti-inflammative effect and reduced cell damage from oxidative stress, thus could be applied to the treatment or prevention of AMD.

目錄............................................................................................................................I
附圖目錄................................................................................................................IV
縮寫表.......................................................................................................................I
中文摘要..................................................................................................................1
Abstract....................................................................................................................3
第一章【前言】.................................................................................5
一、 老年性黃斑部病變......................................................................5
二、 TNF-α...........................................................................................6
三、 NF-κB...........................................................................................7
四、 茄紅素 (lycopene).......................................................................8
五、 Nrf-2.............................................................................................9
六、 GSH............................................................................................10
七、 研究目的....................................................................................12
第二章【實驗材料及方法】........................................13
一、 實驗藥品 (Chemicals)：...............................................................13
二、 細胞繼代培養 (Cells subculture)：..............................................13
三、 細胞存活率實驗 (Cell viability assay)：.....................................15
四、 傷口癒合細胞轉移測試 ( Wound healing migration assay )：....16
五、 細胞黏附因子測試 ( Co-culture and in vitro monocyte adhesion assay )：.........................................................................................17
六、 RNA萃取 (RNA extraction) ........................................................18
七、 Reverse transcriptase polymerase chain reaction (RT-PCR) ........19
八、 Small interfering RNA (siRNA) ...................................................22
九、 總蛋白質萃取及定量 (Preparation of whole cell lysates and protein quantification) ...................................................................23
十、 細胞核內蛋白質萃取 (Preparation of nuclear lysates)：............25
十一、 西方墨點法 (Western blotting)：.............................................27
十二、 細胞內glutathione (GSH)含量測試 (GSH assay)：...............30
十三、 細胞抗氧化能力測試 (Cell antioxidant ability assay)：.........31
十四、 細胞內ROS含量測試：..........................................................32
十五、 質體轉染和啟動子活性試驗 (transfection and promoter assay)： .......................................................................................33
十六、 資料分析 (Statistical analysis)：.............................................34
第三章【實驗結果】........................................................35
一、 細胞茄紅素對人類視網膜色素上皮細胞的毒性測試................35
二、 茄紅素對視網膜色素上皮細胞移行能力的作用........................35
三、 茄紅素抑制單核球THP-1黏附至TNF-α所活化之視網膜色素上皮細胞...........................................................................................35
四、 茄紅素抑制視網膜色素上皮細胞ICAM-1蛋白質的表現.........36
五、 茄紅素抑制視網膜色素上皮細胞因TNF-α所誘導之轉錄因子NF-κB-p65轉位及轉錄活性........................................................36
六、 茄紅素影響視網膜色素上皮細胞對TNF-α所誘導之IκB降解情況....................................................................................................37
七、 茄紅素增加Nrf-2的轉位入核效率並活化Nrf-2的基因表現......38
八、 茄紅素增加細胞內Glutathione的表現量.....................................39
九、 茄紅素降低TNF-α造成的氧化壓力............................................39
十、 茄紅素減少氧化壓力造成的視網膜色素上皮細胞損傷...........40
第四章【討論】..........................................................41
第五章【總結】..........................................................45
附圖...............................................................................46
References.....................................................................62

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