PEDF and GDNF are key regulators of photoreceptor development and retinal neurogenesis in reaggregates from chick embryonic retina.

Katja N Volpert, Joyce Tombran-Tink, Colin Barnstable, Paul G Layer
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引用次数: 24

Abstract

Here, role(s) of pigment epithelial-derived factor (PEDF) and glial-derived neurotrophic factor (GDNF) on photoreceptor development in three-dimensional reaggregates from the retinae of the E6 chick embryo (rosetted spheroids) was investigated. Fully dispersed cells were reaggregated under serum-reduced conditions and supplemented with 50 ng/ml PEDF alone or in combination with 50 ng/ml GDNF. The spheroids were analyzed for cell growth, differentiation, and death using proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, and other immunocytochemical stainings and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) methods. PEDF strongly promoted synthesis of the messenger RNAs for blue and violet cone opsins and to a lesser extent on the red and green cone opsins. This correlated with an increase in the number of cone photoreceptors, as determined by the cone cell marker CERN906. Likewise, PEDF nearly completely inhibited rod differentiation, as detected by immunostaining with anti-rho4D2 and RT-PCR. Furthermore, PEDF accelerated proliferation of cells in the spheroids and inhibited apoptosis. As negative effects, PEDF inhibited the normal histotypic tissue formation of retinal aggregates and reduced the frequency of photoreceptor rosettes and IPL-like areas. Noticeably, supplementation of PEDF-treated cultures with GDNF reversed the effects of PEDF on spheroid morphology and on rod differentiation. This study establishes that PEDF strongly affects three-dimensional retinogenesis in vitro, most notably by inhibiting rod development and supporting proliferation and differentiation of cones, effects which are partially counteracted by GDNF.

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PEDF和GDNF是鸡胚视网膜再聚体光感受器发育和视网膜神经发生的关键调节因子。
本研究研究了色素上皮衍生因子(PEDF)和胶质衍生神经营养因子(GDNF)在E6鸡胚胎视网膜三维再聚体(玫瑰球状体)光感受器发育中的作用。充分分散的细胞在血清减少的条件下重新聚集,并单独添加50 ng/ml PEDF或与50 ng/ml GDNF联合。采用增殖细胞核抗原、末端脱氧核苷酸转移酶三磷酸脱氧尿苷缺口端标记、其他免疫细胞化学染色和半定量逆转录聚合酶链反应(RT-PCR)方法分析球体细胞的生长、分化和死亡情况。PEDF强烈促进蓝色和紫色视锥蛋白信使rna的合成,对红色和绿色视锥蛋白的作用较小。这与视锥细胞标记物CERN906确定的视锥光感受器数量的增加有关。同样,通过抗rho4d2和RT-PCR免疫染色检测,PEDF几乎完全抑制棒分化。此外,PEDF加速了球体细胞的增殖并抑制了细胞凋亡。作为负面影响,PEDF抑制了视网膜聚集体的正常组织形成,减少了光感受器莲座和ipl样区域的频率。值得注意的是,用GDNF补充PEDF处理过的培养物逆转了PEDF对球体形态和杆分化的影响。本研究证实,PEDF强烈影响体外三维视网膜形成,最明显的是通过抑制杆发育和支持视锥细胞的增殖和分化,这些作用部分被GDNF抵消。
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