- 學位論文
以動物及細胞培養模式探討LED燈對於視網膜感光細胞潛藏損傷
The progression of photoreceptor cell damage by low-powered LED illumination in vivo and in vitro
摘要
利用人類及動物實驗研究已經證實，照射過量可見光會對眼部組織造成損傷。但是，波長範圍介於430 nm至500 nm（藍光區域）短波常可見光，則是無法被角膜及水晶體吸收，對視網膜損傷特別有關連性。近年來，一種新的能量來源及相對較便宜的照明LED，已被廣泛使用於家庭照明之中。然而，低功率LED照明對於視網膜感光細胞造成的損傷目前尚未釐清。本研究主要之目的為，探討居家常用低功率LED對於視網膜之影響。為了了解低功率LED照射對於視網膜的影響，本研究使用常見黑色小鼠C57BL/6及人類視網膜母細胞瘤 Y79細胞株進行LED照射，並進行後續評估。利用測定波長範圍300 nm至800 nm的UV/可見光光譜儀測定居家常用之低功率LED之波長範圍。動物實驗中，我們將小鼠分為每天利用家用LED光照2小時持續2週到4週以及持續性環境光照39週。在不同時間點LED光線照射後，將動物犧牲，取其眼球進行組織病理學分析。我們利用組織病理學及細胞凋亡試劑TUNEL染色，檢測LED光線照射及細胞死亡之間的關係。組織病理學結果表示，在LED照射下，確實會對視網膜造成傷害，感光細胞層的厚度在光照每天2小時持續4週及持續性環境光照之下有顯著性的變薄。LED光線照射後，感光細胞層確實是明顯的厚度減少，並且可以觀察到在感光細胞層的部分有TUNEL接合反應的表現。體外細胞培養實驗中，人類視網膜母細胞瘤Y79細胞同樣也是利用LED光線照射持續1、2、4天。光線照射後對細胞進行形態學、免疫化學染色及西方墨點法分析。在LED光照後，可以觀察到細胞型態有明顯的變小，並且出現細胞核濃染以及細胞凋亡的型態。接著利用免疫染色及西方墨點法發現，DNA修復蛋白p53及細胞凋亡標記蛋白caspase-3的表現會隨著光照時間的增加而有變多的趨勢。證實了細胞經過LED光線照射後確實是走向細胞凋亡之路徑。本研究實驗結果對於實驗動物小鼠模式照射居家常用低功率LED照明之有效性及安全性上提供一個重要的實驗參考數據。
並列摘要
Ocular tissue damage because of exposure to visible light has been demonstrated by the results of human and animal studies. The short-wavelength visible light between 430 nm to 500 nm（blue light）is especially associated with retina damage. Recently, new powerful sources and relatively inexpensive blue energy of LED（light emitting diodes）family lamps in home illumination are available. However, the retinal photoreceptor damage produced by the low-powered LED illumination was not addressed. The aim of this study is to investigate the effects of illumination source from the low-powered and the conscious spectrum source of LED family lamps on retina. In order to investigate the influence of low-powered LED illumination on retina, LED emission was examined in C57BL/6 mice and retinoblastoma Y79 cells. The LED light was analyzed from 300nm to 800nm using an UV-visible spectrophotometry. In vivo, young adult mice were assigned to expose to family LED light for 2hours every day ranging 2 to 4 weeks or light environment using LED family lamps for 39 weeks. At different times after light exposure, animals were sacrificed and eyeballs were dissected as cross-sections. To illustrate the relationship between LED exposure and cell death, the histopathology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling（TUNEL）assay were applied. Morphological results showed that the photoreceptor layer is significantly reduced in thickness after 4 weeks of LED exposure 2hours every day or LED illuminated environment. After light exposure, the photoreceptor layer was significantly reduced in thickness and TUNEL-positive nuclei were detected in the photoreceptor layer. In vitro, the human retinoblastoma cell line Y79 was also irradiated with LED light ranging 1、2 to 4 days. Cultured cells were analyzed by morphology, immunocytochemistry, and Western blot analysis. The cell stage of Y79 cell was smaller than that of the control, and the apoptosis-like characteristic of nuclei condensation could be observed. Increased p53 and caspase-3 proteins were detected in Y79 cells after light exposure. This study provides important information regarding the efficacy and safety in animal models of LED light in family illumination.
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