Lu Fan, Yan Dongsheng, Zhou Xiangtian, Hu Dan-Ning, Qu Jia
Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical College and Key Laboratory of Vision Science, Ministry of Health PR China, Wenzhou China.
Mol Vis. 2007 Nov 3;13:2066-72.
Controversy exists over melanogenesis of adult human RPE cells in vitro. This study investigated melanin content and production and expression of tyrosinase (TYR), tyrosinase-related-protein-1 (TRP1), tyrosinase-related-protein-2 (TRP2), and P gene in cultured human RPE cells and uveal melanoma cells.
RPE cells were isolated and cultured from three adult donor eyes. A continuous human uveal melanoma cell line was established from primary choroidal melanoma. Melanin content and production were measured, and the expression of TYR, TRP1, TRP2, and P gene at the mRNA and protein levels were detected by RT-PCR and western blot, respectively.
Melanin content per cell of cultured RPE decreased rapidly and in proportion to cell division. No melanin production could be demonstrated in any passages. In cultured RPE cells, mRNA expression of TYR, TRP1, TRP2, and P-gene and protein expression of TYR, TRP1, and TRP2 could not be detected. In uveal melanoma cells, melanin content per cell remained stable, and melanin production could be detected in each passage. Expression of mRNA of TYR, TRP1, TRP2, and P-gene and protein of TYR, TRP1, and TRP2 could be detected in melanoma cells.
Human RPE cells under standard culture circumstances do not produce melanin and do not express the four key genes required in melanin biosynthesis pathway. In contrast, human uveal melanoma cells produce melanin and express all of these melanogenic genes in vitro.
体外培养的成人视网膜色素上皮（RPE）细胞的黑色素生成存在争议。本研究调查了培养的人RPE细胞和葡萄膜黑色素瘤细胞中黑色素含量、生成情况以及酪氨酸酶（TYR）、酪氨酸酶相关蛋白-1（TRP1）、酪氨酸酶相关蛋白-2（TRP2）和P基因的表达。
从三只成人供体眼中分离并培养RPE细胞。从原发性脉络膜黑色素瘤建立连续的人葡萄膜黑色素瘤细胞系。测量黑色素含量和生成情况，并分别通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测TYR、TRP1、TRP2和P基因在mRNA和蛋白质水平的表达。
培养的RPE细胞中每个细胞的黑色素含量迅速下降，且与细胞分裂成比例。在任何传代中均未显示有黑色素生成。在培养的RPE细胞中，未检测到TYR、TRP1、TRP2和P基因的mRNA表达以及TYR、TRP1和TRP2的蛋白质表达。在葡萄膜黑色素瘤细胞中，每个细胞的黑色素含量保持稳定，并且在每个传代中都能检测到黑色素生成。在黑色素瘤细胞中可检测到TYR、TRP1、TRP2和P基因的mRNA表达以及TYR、TRP1和TRP2的蛋白质表达。
在标准培养条件下，人RPE细胞不产生黑色素，也不表达黑色素生物合成途径所需的四个关键基因。相比之下，人葡萄膜黑色素瘤细胞在体外产生黑色素并表达所有这些黑色素生成基因。
