吳啟豪Wu, Chi-Hao張瑀宸Chang, Yu-Chen2020-10-192023-09-152020-10-192020http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22G060751012S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/111988中華民國眼科醫學會最新統計 (2020)，國人每日平均使用3C設備 (Computer, Communications, and Consumer-Electronics)時間長達10小時。已知眼睛長期暴露於含藍光之LED (light-emitting diode)發光源下，易引發視網膜病變，造成視力下降，因此尋求具有減緩藍光損傷之護眼機能性成分，於維護視力健康上有其重要意義。生理高血糖與高血脂為代謝症候群之共同特徵，可造成體內氧化壓力與慢性發炎反應，亦可能為加劇藍光氧化損傷之惡化因子，因此本研究擬針對上述兩項主題進行探討：第一部分利用藍光合併光敏感物質A2E (N-retinylidene-N-retinylethanolamine)誘導人類視網膜色素上皮細胞株ARPE-19損傷為模式，嘗試從天然植化素中篩選具有減低藍光損傷之機能性成分。首先將all-trans-retinol與ethanolamine混合反應以進行A2E化學合成，反應物經silica gel-C18 based column chromatography及cation exchange chromatography純化後，以1H-NMR及LC-MS/MS進行A2E結構鑑定。結果顯示，A2E於生理濃度範圍 (6–25 μM)內，經藍光照射9–24 h後，均可顯著造成ARPE-19細胞株之細胞毒性；篩選植化素樣品中，以quercetin、morin、resveratrol及grcinol具有減輕藍光損傷之保護效果；其中尤以quercetin活性最佳，作用劑量20 μM即具有52%保護率。論文第二部分乃探討葡萄糖與脂肪酸是否為影響藍光損傷之可能因子，實驗模擬正常生理血糖濃度5.0 mM (90 mg/dL)與病理性高血糖濃度17.5 mM (315 mg/dL)及30 mM (540 mg/dL)為模式，測試ARPE-19細胞於不同葡萄糖濃度培養下，經藍光照射後對細胞存活率之影響。結果顯示，有別於正常葡萄糖濃度，當細胞培養於17.5 mM或30 mM高糖濃度時可顯著提高藍光對ARPE-19細胞之細胞毒性，此顯示高糖環境可能為促進藍光損傷之惡化因子。此外，已知棕櫚酸 (palmitic acid, PA)、硬脂酸 (stearic acid, SA)、亞麻油酸 (linoleic acid, LA)和花生四烯酸 (arachidonic acid, AA)為高血脂患者體內濃度較高之四種脂肪酸；同時，二十二碳六烯酸 (docosahexaenoic acid, DHA)為視網膜中含量最豐富的脂肪酸，因此本研究進一步探討ARPE-19細胞在不同葡萄糖濃度 (5與17.5 mM)下，分別與上述5種脂肪酸共培養後，再暴露於藍光下之細胞毒性變化。結果顯示，無論有無藍光照射，ARPE-19細胞與飽和脂肪酸PA或SA培養24 h後，其細胞存活率與控制組比較均無顯著影響 (p > 0.05)。有趣的是，不飽和程度較高之AA與DHA可顯著加劇藍光對ARPE-19細胞之細胞毒性，此顯示藍光可能引發不飽和脂肪酸之脂質過氧化作用，致使視網膜色素上皮細胞死亡。綜合上述，本研究發現多酚類化合物quercetin、morin、resveratrol及grcinol具有保護視網膜色素上皮細胞免於藍光氧化損傷之潛力；病理性高葡萄糖濃度與多元不飽和脂肪酸AA及DHA可能為加劇藍光氧化損傷之促進因子，未來有待進一步體內試驗釐清與驗證。The Ophthalmological Society of Taiwan (2020) reported that people spend ten hours using electronic products of daily life. Exposure to blue light for prolonged periods of time cause retinopathy and vision lose. It is known that hyperglycemia and hyperlipidemia are common features of metabolic syndrome, which will cause increased oxidative stress and chronic inflammation. This study aims to investigate the effects of blue light on ARPE-19 cell toxicity under high glucose and high fatty acid conditions. There are two topics in the thesis, and these topics are described as follows: The first topic is to use human retinal epithelial cell line ARPE-19 and to select functional components that can prevent the damaged of blue light irradiation combined with A2E. First, we synthesize A2E by all-trans-retinol and ethanolamine, then purified A2E by silica gel-C18 based column chromatography and cation exchange chromatography and identified A2E structure by 1H-NMR and LC-MS/MS. The results have shown that A2E in the physiological concentration of 6 – 25 μM combined with blue light irradiation 9–24 hours significantly induce cytotoxicity. Among the polyphenols that we screened, quercetin, morin, resveratrol and grcinol can reduce blue light induce oxidative damage. The second topic is to investigate whether the different blood glucose and blood lipid concentrations are aggravating factors of blue light damage. We use normal physiological glucose concentration of 5 mM (90 mg / dL) and pathological hyperglycemia concentration of 17.5 mM (315 mg / dL) and 30 mM (540 mg / dL), tested its cytotoxicity of ARPE-19 cell after irradiation of blue light 24 hours. The results have shown that, unlike the normal blood glucose concentration, when cells are cultured at high glucose concentrations of 17.5 and 30 mM, the toxicity of blue light to ARPE-19 cells can be significantly aggravated. It is known that palmitic acid, stearic acid, LA and arachidonic acid are the 4 fatty acids with higher concentration in patients with hyperlipidemia. DHA in the most abundant fatty acid in the retina, so we further investigate ARPE-19 cells under different glucose concentrations (5, 17.5 mM), co-cultured with the above five different fatty acids, under blue light irradiation.The results have shown that ARPE-19 cells were incubated with palmitic acid or stearic acid for 24 hours with or without blue light irradiation, and had no significant effect on cell viability (p > 0.05). It is worth noting that it will increase the cytotoxicity of unsaturated fatty acids (UFA) to ARPE-19 cell when unsaturated fatty acids combined with blue light irradiation. While blue light irradiation may cause UFA peroxidation, causing retinal cell damage or apoptosis. In conclusion, this study found that supplementation with quercetin, morin, resveratrol, and grcinol may protect the eyes from photooxidative damage. At pathologically high blood glucose concentrations, DHA and AA, it may exacerbate the cytotoxicity of blue light irradiation combined with A2E that to be clarified and verified by animal experiments in the future.藍光葡萄糖視網膜色素上皮細胞A2E多酚類化合物脂肪酸A2Eblue lightRPE cellglucosefatty acid