PDF(Pubmed)
Abstract：
Interaction between retinal vascular endothelial cells and neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR). This study aims to compare an in vitro model over a monoculture model to simulate the neurovascular coupling under the hyperglycemic microenvironment of diabetes.
Rat retinal vascular endothelial cells (RRMECs) and ganglion cells (RGCs) were seeded mono- or co-cultured in a normal (NG, 5.5 mM) and high (HG, 75 mM) glucose concentrations culture medium. Cell viability was detected by the cell counting kit-8 (CCK-8) assay. The ability of migration and lumen formation of RRMECs were determined by scratch wound, transwell migration, and lumen formation assays. The apoptosis index of cells was calculated and detected by propidium iodide (PI)/Hoechst staining. Quantitative and morphological analysis of RGCs was performed through the labeling of RGCs by brain-specific homeobox/POU domain protein 3A (BRN3A) and anti-beta-III tubulin (TUJ1). The gene and protein expression levels of occludin (OCLN) and zonula occludens-1 (ZO-1) were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.
The viability, migration, and lumen formation abilities of RRMECs in the HG group significantly increased (P<0.05) in both mono- and co-culture models. Migration and lumen formation abilities of RRMECs in the co-culture with HG were lower than that in the monoculture group (P<0.05). The viability of RGCs cells with HG significantly decreased in both mono- and co-culture models (Pmono<0.001, Pco<0.001), the apoptosis index of RGCs in the co-culture with HG was higher than that in the monoculture (P=0.010). The protein and gene expression of OCLN, and ZO-1 in RRMECs significantly decreased with HG culture medium in both culture models (P<0.05). In the HG group, the protein and gene expression level of the ZO-1 and OCLN of RRMECs significantly decreased in the co-culture model than that in the monoculture model (P<0.05).
Compared with mono cell culture, the established co-culture in vitro system for diabetic neurovascular dysfunction can better stimulate the micro-environment of the retinal neurovascular unit.
Rat retinal vascular endothelial cells (RRMECs) and ganglion cells (RGCs) were seeded mono- or co-cultured in a normal (NG, 5.5 mM) and high (HG, 75 mM) glucose concentrations culture medium. Cell viability was detected by the cell counting kit-8 (CCK-8) assay. The ability of migration and lumen formation of RRMECs were determined by scratch wound, transwell migration, and lumen formation assays. The apoptosis index of cells was calculated and detected by propidium iodide (PI)/Hoechst staining. Quantitative and morphological analysis of RGCs was performed through the labeling of RGCs by brain-specific homeobox/POU domain protein 3A (BRN3A) and anti-beta-III tubulin (TUJ1). The gene and protein expression levels of occludin (OCLN) and zonula occludens-1 (ZO-1) were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.
The viability, migration, and lumen formation abilities of RRMECs in the HG group significantly increased (P<0.05) in both mono- and co-culture models. Migration and lumen formation abilities of RRMECs in the co-culture with HG were lower than that in the monoculture group (P<0.05). The viability of RGCs cells with HG significantly decreased in both mono- and co-culture models (Pmono<0.001, Pco<0.001), the apoptosis index of RGCs in the co-culture with HG was higher than that in the monoculture (P=0.010). The protein and gene expression of OCLN, and ZO-1 in RRMECs significantly decreased with HG culture medium in both culture models (P<0.05). In the HG group, the protein and gene expression level of the ZO-1 and OCLN of RRMECs significantly decreased in the co-culture model than that in the monoculture model (P<0.05).
Compared with mono cell culture, the established co-culture in vitro system for diabetic neurovascular dysfunction can better stimulate the micro-environment of the retinal neurovascular unit.
摘要：
■视网膜血管内皮细胞与神经元之间的相互作用在糖尿病视网膜病变（DR）的发病机制中起着至关重要的作用。本研究旨在比较体外模型与单一培养模型，以模拟糖尿病高血糖微环境下的神经血管偶联。
■将大鼠视网膜血管内皮细胞（RRMEC）和神经节细胞（RGCs）在正常（NG，5.5mM）和高（HG，75mM)葡萄糖浓度培养基。通过细胞计数试剂盒-8(CCK-8)测定检测细胞活力。通过划痕确定RRMEC的迁移和管腔形成能力，Transwell迁移,和管腔形成测定。计算细胞凋亡指数并通过碘化丙啶(PI)/Hoechst染色检测。通过用脑特异性同源异型盒/POU结构域蛋白3A（BRN3A）和抗β-III微管蛋白（TUJ1）标记RGC，对RGC进行定量和形态学分析。通过定量实时聚合酶链反应和酶联免疫吸附测定评估了闭塞蛋白（OCLN）和闭塞带1（ZO-1）的基因和蛋白质表达水平。
■生存能力，迁移,在单培养和共培养模型中，HG组RRMEC的管腔形成能力显着增加（P<0.05）。与HG共培养的RRMECs的迁移和管腔形成能力均低于单一培养组（P<0.05）。在单培养和共培养模型中，具有HG的RGC细胞的活力显着降低（Pmono<0.001，Pco<0.001），与HG共培养的RGCs的凋亡指数高于单一培养的RGCs（P=0.010）。OCLN的蛋白和基因表达,在两种培养模型中，RRMECs中的ZO-1和HG培养基均显着降低（P<0.05）。在HG组中,共培养模型中RRMECs的ZO-1和OCLN的蛋白和基因表达水平明显低于单一培养模型(P<0.05)。
■与单细胞培养相比，建立的糖尿病神经血管功能障碍共培养体外系统可以更好地刺激视网膜神经血管单元的微环境。
■将大鼠视网膜血管内皮细胞（RRMEC）和神经节细胞（RGCs）在正常（NG，5.5mM）和高（HG，75mM)葡萄糖浓度培养基。通过细胞计数试剂盒-8(CCK-8)测定检测细胞活力。通过划痕确定RRMEC的迁移和管腔形成能力，Transwell迁移,和管腔形成测定。计算细胞凋亡指数并通过碘化丙啶(PI)/Hoechst染色检测。通过用脑特异性同源异型盒/POU结构域蛋白3A（BRN3A）和抗β-III微管蛋白（TUJ1）标记RGC，对RGC进行定量和形态学分析。通过定量实时聚合酶链反应和酶联免疫吸附测定评估了闭塞蛋白（OCLN）和闭塞带1（ZO-1）的基因和蛋白质表达水平。
■生存能力，迁移,在单培养和共培养模型中，HG组RRMEC的管腔形成能力显着增加（P<0.05）。与HG共培养的RRMECs的迁移和管腔形成能力均低于单一培养组（P<0.05）。在单培养和共培养模型中，具有HG的RGC细胞的活力显着降低（Pmono<0.001，Pco<0.001），与HG共培养的RGCs的凋亡指数高于单一培养的RGCs（P=0.010）。OCLN的蛋白和基因表达,在两种培养模型中，RRMECs中的ZO-1和HG培养基均显着降低（P<0.05）。在HG组中,共培养模型中RRMECs的ZO-1和OCLN的蛋白和基因表达水平明显低于单一培养模型(P<0.05)。
■与单细胞培养相比，建立的糖尿病神经血管功能障碍共培养体外系统可以更好地刺激视网膜神经血管单元的微环境。
