不同浓度A2E对离体猪视网膜色素上皮细胞的影响

目的评价体外合成的A2E对猪视网膜色素上皮（RPE）细胞的细胞活力和生物学特性影响，为进一步研究A2E在RPE细胞相关疾病中的作用提供细胞模型。方法利用全反式视黄醛和乙醇胺体外合成脂褐质荧光基团A2E。不同浓度的A2E（0，50，75，100μmol／L）作用第3代体外培养的猪RPE细胞30，45，60，90min，换10％FBS DMEM-F12培养液孵育24h后，倒置荧光显微镜观察荧光强度，IPP6．0软件灰度扫描定量荧光强度。采用MTT法检测A2E作用细胞各个时间段的吸光度值，应用SPSS11．0软件包对数据进行统计学分析，评价A2E的细胞毒性及RPE细胞活性。结果A2E被RPE细胞摄取后主要分布于细胞核周围，具有自发荧光。MTT实验及荧光灰度扫描结果显示，不同浓度的A2E被细胞摄取后细胞活力和荧光灰度扫描结果不同，以50μmol／L浓度A2E作用RPE细胞60min时，细胞内荧光强度高同时细胞活力强。结论体外培养的猪RPE细胞摄取体外合成的50μmol／L A2E 60min后细胞对A2E的摄取较多，A2E对细胞的毒性相对较低，该条件下进行A2E对离体猪RPE细胞的研究较好。

Effect of A2E at Different Concentrations on Pig Retinal Pigment Epithelium in vitro

Objective To observe the cell activity and biological characteristics of retinal pigment epithelium （RPE） cells in vitro after internalization of A2E and establish a cell model for research on A2E related RPE disease. Methods All-transretinal and triethanolamine were used for synthesis of lipofuscin fluorophore A2E in vitro. The solution of TLC （Thin Layer Chromatographic Separation ）-purified A2E in methanol was subjected to room temperature and light. A2E with different concentrations （50,75,100 μmol/L） were delivered to cultured RPE cells in media for internalization. Cells were observed under fluorescence microscope at 30,45,60,90 minutes and MTT assay was used to detect the viable cells. Results When A2E was delivered to RPE cells in culture, it accumulated intracellularly. Internalized A2E presented as autofluorescent granules having a perinuclear distribution. MTT assay and grayscale scanning fluorescence results show that variable cells to different degrees correspond at different concentrations of A2E uptake by the cells and 50 μmol/L in 60 minutes was the best experimental condition. Conclusions The best condition for experiment on A2E and pig RPE cells is the concentration of 50 μmol/L and the time of 60 minutes.