North Texas Eye Research Institute, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, United States.
North Texas Eye Research Institute, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, United States.
Exp Eye Res. 2018 Nov;176:188-195. doi: 10.1016/j.exer.2018.07.011. Epub 2018 Jul 10.
Optic nerve astrocytes play a major role in axonal degeneration and regeneration. Astrocyte lines are an important tool to elucidate the responsible cellular mechanisms. In this study, we established a conditionally immortalized mouse optic nerve astrocyte line. Astrocytes were cultured from explants derived from postnatal day 4-5 H-2k-tsA58 transgenic mouse optic nerves. Cells were cultured in defined astrocyte culture medium under permissive (33 °C) or non-permissive (38.5 °C) temperatures with or without interferon-ɤ (IFN-ɤ). Astrocytes were characterized by immunocytochemistry staining using antibodies against glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (NCAM). Cell proliferation rates were determined by cell growth curves and percentage of Ki67 positive cells. Karyotyping was performed to validate the mouse origin of established cell line. Conditional immortalization was assessed by western blot-determined expression levels of SV40 large T antigen (TAg), p53, GFAP and NCAM in non-permissive culture conditions. In addition, phagocytic activity of immortalized cells was determined by flow cytometry-based pHrodo fluorescence analysis. After 5 days in culture, cells migrated out from optic nerve explants. Immunocytochemistry staining showed that migrating cells expressed astrocyte makers, GFAP and NCAM. In permissive conditions, astrocytes had increased expression levels of TAg and p53, exhibited a greater cell proliferation rate as well as a higher percentage of Ki67 positive cells (n = 3, p < 0.05) compared to cells cultured in non-permissive conditions. One cell line (ImB1ON) was further maintained through 60 generations. Karyotyping showed that ImB1ON was of mouse origin. Flow cytometry-based pHrodo fluorescence analysis demonstrated phagocytic activity of ImB1ON cells. Quantitative PCR showed mRNA expression of trophic factors. Non-permissive culture conditions decreased expression of TAg and p53 in ImB1ON, and increased the expression of NCAM. A conditionally immortalized mouse optic nerve astrocyte line was established. This cell line provides an important tool to study astrocyte biological processes.
视神经星形胶质细胞在轴突变性和再生中起主要作用。星形胶质细胞系是阐明相关细胞机制的重要工具。在本研究中，我们建立了一种条件永生化的小鼠视神经星形胶质细胞系。星形胶质细胞是从出生后 4-5 天 H-2k-tsA58 转基因小鼠视神经衍生的外植体中培养出来的。细胞在有或没有干扰素-γ (IFN-γ)的条件下，在允许温度（33°C）或非允许温度（38.5°C）下，在限定的星形胶质细胞培养基中培养。通过针对神经胶质纤维酸性蛋白 (GFAP) 和神经细胞黏附分子 (NCAM) 的免疫细胞化学染色来鉴定星形胶质细胞。通过细胞生长曲线和 Ki67 阳性细胞的百分比来确定细胞增殖率。通过核型分析来验证建立的细胞系的小鼠来源。通过 Western blot 测定非允许培养条件下 SV40 大 T 抗原（TAg）、p53、GFAP 和 NCAM 的表达水平来评估条件永生化。此外，通过基于流式细胞术的 pHrodo 荧光分析来测定永生化细胞的吞噬活性。培养 5 天后，细胞从视神经外植体中迁移出来。免疫细胞化学染色显示迁移细胞表达星形胶质细胞标志物 GFAP 和 NCAM。在允许条件下，与非允许条件下培养的细胞相比，星形胶质细胞的 TAg 和 p53 表达水平增加，细胞增殖率更高，Ki67 阳性细胞的百分比更高（n=3，p<0.05）。一条细胞系（ImB1ON）进一步维持了 60 代。核型分析表明 ImB1ON 来源于小鼠。基于流式细胞术的 pHrodo 荧光分析表明 ImB1ON 细胞具有吞噬活性。定量 PCR 显示营养因子的 mRNA 表达。非允许培养条件降低了 ImB1ON 中 TAg 和 p53 的表达，并增加了 NCAM 的表达。建立了一种条件永生化的小鼠视神经星形胶质细胞系。该细胞系为研究星形胶质细胞的生物学过程提供了重要工具。
