Sheedlo H J, Krishnamoorthy R S, Nelson T S, Agarwal N S, Liu J S, Roque R S, Wordinger R J, Jaynes C D, Brun-Zinkelnagel A M, O'Brian P, Aschenbrenner J E, Turner J E
University of North Texas Health Science Center, North Texas Eye Research Institute, Department of Anatomy and Cell Biology, Fort Worth, TX 76107, USA.
Curr Eye Res. 1999 Dec;19(6):465-71. doi: 10.1076/ceyr.19.6.465.5274.
This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light micros-copy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot.
Retinal tissues and sections from embryonic, early postnatal and adult normal rats and retinal pigment epithe-lial (RPE) cells from postnatal rats were immunostained for fetuin with a polyclonal fetuin antibody and a peroxidase conjugated-secondary antibody using immunocytochemistry and Western blot analyses. The cDNA generated from RNA isolated from early postnatal rat retinas and RPE was probed with primers for rat fetuin, amplified by PCR and the PCR products were analyzed by Southern blot.
Fetuin (RDF) was immunolocalized to cells of the neuroepithelium in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-PCR, the message for rat fetuin was demonstrated in the retina and RPE of normal postnatal rats. Southern blot confirmed that the PCR product from the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin.
Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.
本研究旨在确定视网膜衍生胎球蛋白（RDF）蛋白及其信使核糖核酸在视网膜组织和视网膜色素上皮（RPE）细胞中的存在情况。本研究采用的技术包括光学显微镜检查、免疫化学、蛋白质印迹法、逆转录聚合酶链反应（RT-PCR）和Southern印迹法。
使用免疫细胞化学和蛋白质印迹分析，用多克隆胎球蛋白抗体和过氧化物酶偶联二抗对来自胚胎期、出生后早期和成年期正常大鼠的视网膜组织和切片以及出生后大鼠的视网膜色素上皮（RPE）细胞进行胎球蛋白免疫染色。用大鼠胎球蛋白引物探测从出生后早期大鼠视网膜和RPE分离的RNA产生的互补脱氧核糖核酸（cDNA），通过聚合酶链反应（PCR）扩增，并用Southern印迹法分析PCR产物。
胎球蛋白（RDF）免疫定位在出生后早期大鼠视网膜的神经上皮细胞中，在培养的新生大鼠RPE细胞的细胞核和核周区域最为明显。在成年视网膜中，神经节细胞、光感受器细胞的内节、外丛状层的一些成分、神经节细胞和视神经突起对胎球蛋白蛋白呈免疫反应性。蛋白质印迹显示，胎球蛋白（RDF）在胚胎期和出生后早期视网膜中比出生后晚期视网膜中含量更高，表明该蛋白可能受发育调控。使用RT-PCR，在出生后正常大鼠的视网膜和RPE中证实了大鼠胎球蛋白的信使核糖核酸。Southern印迹证实，来自视网膜和RPE的PCR产物是由大鼠胎球蛋白信使核糖核酸以及胎球蛋白的主要来源大鼠肝脏产生的。
首次在视网膜组织中报道了胎球蛋白，称为视网膜衍生胎球蛋白（RDF）。胎球蛋白是一种半胱氨酸蛋白酶抑制剂，可能在视网膜早期发育过程中支持神经元细胞存活以及维持神经元活性方面发挥作用。RDF可能与其他生长因子和细胞因子相互作用，为发育中的视网膜中的神经元以及可能的其他细胞提供营养支持。
