脉络膜全反式维甲酸(atRA)可能通过调节巩膜细胞外基质的合成在多种脊椎动物的出生后眼部生长中起关键作用,因此可能在近视的发展中起关键作用。在小鸡眼中,脉络膜atRA合成完全由其合成酶调节,视黄醛脱氢酶2(RALDH2)。在小鸡和人类中,RALDH2已在迄今未表征的脉络膜细胞群中检测到。因此,本研究的目的是确定脉络膜中的RALDH2+细胞类型,并确定这些细胞在视觉引导的眼部生长期间如何调节atRA浓度.小鸡在一只眼睛上佩戴半透明护目镜10天,并在摘除护目镜后第0、1、4、7、15天分析脉络膜的RALDH活性和RALDH2蛋白表达(“恢复”);对侧眼的脉络膜作为对照。使用多光子显微镜在小鸡脉络膜整体中评估RALDH2细胞的存在。通过蛋白质印迹测量RALDH2蛋白表达,并通过atRA的HPLC定量评估RALDH2活性。通过BrdU标记结合RALDH2-免疫组织化学评估细胞增殖。对于RALDH2+细胞的表征,在鸡中应用了各种组织特异性标记的免疫组织化学(Ia抗原,CD5,Col1-前肽,desmin,IgY,L-Cam,Cadherin1,MHC-II;Tcr-γδ,波形蛋白)和人类供体组织(α-平滑肌肌动蛋白,CD's31/34/68/146,desmin,IBA1,LYVE-1,PGP9.5,波形蛋白),然后进行共聚焦显微镜检查。在小鸡和人类脉络膜中,具有不同形态的RALDH2+细胞存在于基质中和脉络膜血管附近。在小鸡整体中,RALDH2+细胞向脉络膜毛细血管聚集,与相应的对照组相比,它们的数量在恢复的1到7天之间几乎线性增加,在7到15天之间趋于平稳。通过westernblot和HPLC发现,通过4天的回收率(^107%和^120%)观察到脉络膜RALDH2蛋白浓度和atRA合成活性显著增加。分别。与对照组相比,恢复4天后观察到RALDH2/BrDU细胞增加了3倍(恢复眼中所有RALDH2细胞的12.43±0.73%,与对照组眼中的4.46±0.63%相比,p<0.001)。在小鸡脉络膜中,绝大多数RALDH2+细胞共表达Col1-propettide,但没有与任何其他测试的抗体共同标记。在人类脉络膜中,一些,但并非所有的RALDH2+细胞都与波形蛋白共定位,但其他抗体检测均为阴性.RALDH2+细胞代表小鸡和人脉络膜中的新型细胞类型。我们的发现,一些人RALDH2细胞对波形蛋白呈阳性,而所有小鸡RALDH2细胞对Col1呈阳性,这表明RALDH2细胞最类似于血管周围和基质成纤维细胞。恢复4天后,RALDH2/BRDU细胞数量增加表明脉络膜atRA浓度部分受RALDH2细胞增殖控制。这种脉络膜细胞类型的鉴定将提供对负责调节出生后眼部生长的细胞事件的更广泛的理解。并可能为有针对性的近视治疗策略提供新的途径。
Choroidal all- trans -retinoic acid (atRA) may play a key role in the control of postnatal eye growth in a variety of vertebrates through modulation of scleral extracellular matrix synthesis and may therefore play a crucial role in the development of myopia. In the chick eye, choroidal atRA synthesis is exclusively regulated by its synthesizing enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In chicks and humans, RALDH2 has been detected in a population of hitherto uncharacterized choroidal cells.Therefore, the aim of this study was to identify the RALDH2+ cell type(s) in the choroid and determine how these cells modulate atRA concentrations during periods of visually guided eye growth. Chicks wore translucent goggles on one eye for 10 days and choroids were analyzed for RALDH activity and RALDH2 protein expression at days 0, 1, 4, 7, 15 following removal of the goggle (\"recovery\"); choroids from contralateral eyes served as controls. The presence of RALDH2+ cells was assessed in chick choroid wholemounts using multiphoton microscopy. RALDH2 protein expression was measured by western blot and RALDH2 activity was assessed via HPLC quantification of atRA. Cell proliferation was assessed by BrdU-labelling in combination with RALDH2-immunohistochemistry. For characterization of RALDH2+ cells, immunohistochemistry for various tissue specific markers was applied in chicken (Ia antigen, CD5, Col1-propeptide, desmin, IgY, L-Cam, Cadherin1, MHC-II; Tcr-γδ, vimentin) and human donor tissue (α-smooth-muscle-actin, CD\'s 31/34/68/146, desmin, IBA1, LYVE-1, PGP9.5, vimentin) followed by confocal microscopy. In the chick and human choroid, RALDH2+ cells with variable morphology were present in the stroma and adjacent to choroidal blood vessels. In chick wholemounts, RALDH2+ cells were concentrated toward the choriocapillaris, and their number increased nearly linearly between 1 and 7 days of recovery and plateaued between 7 and 15 days compared to corresponding controls. A significant increase in choroidal RALDH2 protein concentration and atRA synthetic activity was observed by four days of recovery (↑107% and ↑120%) by western blot and HPLC, respectively. A 3-fold increase in RALDH2+/BrDU+ cells was observed following 4 days of recovery compared to controls (12.43 ± 0.73% of all RALDH2+ cells in recovering eyes as compared with 4.46 ± 0.63% in control eyes, p < 0.001). In chick choroids, the vast majority of RALDH2+ cells co-expressed Col1-propetide, but did not co-label with any other antibodies tested. In human choroid, some, but not all RALDH2+ cells colocalized with vimentin, but were negative for all other antibodies tested. RALDH2+ cells represent a novel cell type in the chick and human choroid. Our findings that some human RALDH2+ cells were positive for vimentin and all chick RALDH2+ cells were positive for Col1, suggest that RALDH2+ cells most closely resemble perivascular and stromal fibroblasts. The increased number of RALDH2+/BRDU+ cells following 4 days of recovery suggests that choroidal atRA concentrations are partially controlled by proliferation of RALDH2+ cells. The identification of this choroidal cell type will provide a broader understanding of the cellular events responsible for the regulation of postnatal ocular growth, and may provide new avenues for specifically targeted strategies for the treatment of myopia.