目的：玻璃膜疣主要成分胆固醇对人视网膜色素上皮细胞ARPE-19中细胞膜钙ATP酶1(plasma membrane Ca2+ ATPase 1，PMCA1)、L型电压依赖性钙离子通道(L-type voltage-dependent calcium channel，LVDCC)和细胞膜钠钙交换蛋白1(sodium calcium exchange protein 1，NCX1)表达的影响。方法：体外培养ARPE-19细胞，将细胞分为对照组和胆固醇处理组(2.5 mg/mL)，取样时间为0、6、12、24、48、72 h。通过实时定量PCR检测PMCA1、LVDCC和NCX1 mRNA的表达水平，用蛋白质印迹法检测蛋白质的表达水平。结果：主要负责细胞内钙离子外排的PMCA1的mRNA和蛋白表达水平在胆固醇处理下出现下调。在胆固醇处理下，钙流入通道LVDCC和钙稳态调控蛋白NCX1的mRNA和蛋白表达明显增多，并且呈现时间依赖性，都是在24 h或48 h表达最多后出现回落。其中LVDCC表达上调倍数较大。结论：玻璃膜疣主要成分胆固醇可以影响人视网膜色素上皮细胞中钙转运通道蛋白的表达，PMCA1的表达受到胆固醇抑制， LVDCC和NCX1的表达受到胆固醇处理上调。这可能会影响细胞内钙离子外排，引起钙离子内流，是否能进一步导致细胞内钙超载而引起细胞凋亡，值得探讨。
Objective: To study the effects of cholesterol, the main component of drusen, on the expression plasma membrane Ca2+ ATPase 1 (PMCA1), L-type voltage-dependent calcium channel (LVDCC) and cell membrane sodium calcium exchange protein 1 (NCX1) of ARPE-19 cells. Methods: The ARPE-19 cell line was cultured in vitro, and the cells were divided into a control group and a cholesterol treatment group (2.5 mg/mL). The treatment time was 0, 6, 12, 24, 48, 72 hours. Real-time quantitative PCR was used to detect the expression of PMCA1, LVDCC and NCX1 at the mRNA level, and western blot was used to detect the expression at the protein level. Results: The mRNA and protein expression levels of PMCA1 which mainly responsible for the efflux of intracellular calcium ions, was down regulated under cholesterol treatment. Meanwhile, the expression of the mRNA and protein of the calcium inflow channel LVDCC and calcium stability regulatory protein NCX1 were significantly increased, and the time-dependency was present, which was up expressed to 24 or 48 h and then fell back. Among them, the LVDCC expression had a large number of times. Conclusion: Cholesterol, the main component of drusen, can affect the expression of calcium channels in human retinal pigment epithelial cells. The expression of PMCA1 was suppressed by cholesterol, and expression of LVDCC and NCX1 were up-regulated with cholesterol treatment, which may affect intracellular calcium efflux then cause calcium influx. Whether it can further cause intracellular calcium overload and cell death is worth exploring.
目的：研究玻璃膜疣主要成分胆固醇对人视网膜色素上皮细胞ARPE-19中金属硫蛋白表达的影响。方法：体外培养ARPE-19细胞，将细胞分为对照组和胆固醇处理组(2.5 mg/mL)，取样时间为0，6，12，24，48，72h。通过实时定量PCR检测hMT1a，hMT2a和hMT3在转录水平的表达，用蛋白质印迹法检测总金属硫蛋白的表达。结果：在转录水平上hMT1a，hMT2a和hMT3受到胆固醇影响mRNA表达上调，且hMT3上调倍数最大；总金属硫蛋白的蛋白表达随着胆固醇处理时间延长明显增多。结论：玻璃膜疣主要成分胆固醇可以上调人视网膜色素上皮细胞中金属硫蛋白的表达，提示金属硫蛋白表达可受到玻璃膜疣形成起始阶段的刺激，其检测是否能用于年龄相关性黄斑变性的早期发现及早期诊断还需深入探讨。
Objective: To study the effects of cholesterol, the main component of drusen, on the expression of metallothionein of ARPE-19 cells. Methods: The ARPE-19 cell line was cultured in vitro, and the cells were divided into a control group and a cholesterol treatment group (2.5 mg/mL). The treatment time was 0, 6, 12, 24, 48, 72 hours. Real- time quantitative PCR was used to detect the expression of hMT1a, hMT2a and hMT3 at the mRNA level, and Western blot was used to detect the expression at the protein level. Results: The mRNA expression of hMT1a, hMT2a and hMT3 were up-regulated by cholesterol and the protein expression of total MTs was increased with cholesterol treatment. Conclusion: Cholesterol, the main component of drusen, can up-regulate the expression of metallothionein in human retinal pigment epithelial cells, suggesting that the expression of metallothionine can be stimulated by the initial stage of drusen formation. However, whether its detection can be used for the early detection and early diagnosis of age-related macular degeneration or not still needs to be further explored.
