Sheedlo H J, Wordinger R J, Fan W, Turner J E
Department of Anatomy and Cell Biology, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth 76107, USA.
Curr Eye Res. 1997 Feb;16(2):116-26. doi: 10.1076/ceyr.16.2.116.5091.
A newly-derived transformed neonatal rat retinal pigment epithelial (tnrRPE) cell line was investigated: for secreted proteins by electrophoresis, and for basic and acidic fibroblast growth factor (FGF) by immunocytochemistry, Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). The FGFR-1 (flg) receptor, which is recognized by aFGF and bFGF, was studied by RT-PCR.
Retinal pigment epithelial (RPE) cells were isolated from 6-day-old pigmented normal Long Evans rats, and became spontaneously transformed after the second passage.
RPE cells at the 5th through 28th passages expressed the epithelial cell marker cytokeratin and cellular retinaldehyde binding protein (CRALBP), an RPE cell marker, but were negative for glial fibrillary acidic protein (GFAP), as shown by immunofluorescence. Secreted proteins of late passage tnrRPE cells were in a narrow molecular weight range of 60-80kDa, while early passage cells exhibited multiple proteins from 20-200kDa. These tnrRPE cells increased by 17-30 fold over a 4-day culture period. At 5th and 28th passage, immunostaining for bFGF and aFGF was dense within nuclei, but light and diffuse within the cytoplasm of transformed RPE cells. As shown by Northern blot, similar levels of message for bFGF were detected in 5th and 30th passage RPE cells. As shown by Northern blot, similar levels of message for bFGF were detected in 5th and 30th passage RPE cells. Furthermore, as shown by RT-PCR, bFGF mRNA was found in freshly isolated and transformed neonatal rat RPE cells. However, the message for FGFR-1(flg) receptor was detected only in the transformed RPE cells.
This study demonstrated a neonatal rat RPE cell line that proliferated rapidly in vitro, expressed high levels of message for hFGF and FGFR-1(flg) receptor, and continued to express RPE-cell characteristics. Importantly, mRNA levels of confluent cultures of these cells were sufficient for bFGF mRNA blot analysis, which eliminates the necessity for PCR and for using excessive numbers of animals for such studies.
研究一种新衍生的转化新生大鼠视网膜色素上皮（tnrRPE）细胞系：通过电泳分析其分泌蛋白，通过免疫细胞化学、Northern印迹和逆转录聚合酶链反应（RT-PCR）检测碱性和酸性成纤维细胞生长因子（FGF）。通过RT-PCR研究可被aFGF和bFGF识别的FGFR-1（flg）受体。
从6日龄有色正常Long Evans大鼠分离视网膜色素上皮（RPE）细胞，传代两次后自发转化。
第5至28代的RPE细胞表达上皮细胞标志物细胞角蛋白和RPE细胞标志物细胞视黄醛结合蛋白（CRALBP），但免疫荧光显示其胶质纤维酸性蛋白（GFAP）呈阴性。传代后期的tnrRPE细胞分泌蛋白分子量范围较窄，为60 - 80kDa，而传代早期细胞呈现20 - 200kDa的多种蛋白。这些tnrRPE细胞在4天培养期内增加了17 - 30倍。在第5代和第28代时，转化RPE细胞核内bFGF和aFGF免疫染色密集，但细胞质内染色浅且弥散。Northern印迹显示，第5代和第30代RPE细胞中bFGF的信使核糖核酸水平相似。此外，RT-PCR显示，在新鲜分离和转化的新生大鼠RPE细胞中发现了bFGF信使核糖核酸。然而，仅在转化的RPE细胞中检测到FGFR-1（flg）受体的信使核糖核酸。
本研究证明了一种新生大鼠RPE细胞系，其在体外快速增殖，表达高水平的hFGF和FGFR-1（flg）受体信使核糖核酸，并持续表达RPE细胞特征。重要的是，这些细胞汇合培养的信使核糖核酸水平足以进行bFGF信使核糖核酸印迹分析，从而无需进行PCR以及为此类研究使用过多动物。
