Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas
Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas
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DOI：
10.3389/fnagi.2016.00093
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发表时间：
2016-05-18
影响因子：
4.8
通讯作者：
Morales-Tirado, Vanessa M.
中科院分区：
文献类型：
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作者：
Chintalapudi, Sumana R.;Djenderedjian, Levon;Morales-Tirado, Vanessa M.
功能性视网膜神经节细胞（RGC）的丧失是视网膜变性的一个要素，但人们对此知之甚少。这部分是由于缺乏用于分离原代RGC的可靠且经验证的方案。在这里，我们优化了一个可行的，可重复的，标准化的流式细胞术为基础的协议，用于分离和富集同质RGC与Thy1.2（高）CD 48（阴性）CD 15（阴性）CD 57（阴性）表面表型。通过以下三步验证过程进行：（1）与视网膜细胞相关的25个基因的基因组谱分析;（2）针对细胞内RGC标记物SNCG、脑特异性同源框/POU结构域蛋白3A（BRN 3A）、TUJ 1和具有多重剪接的RNA结合蛋白（RBPMS）对同质分选的细胞进行细胞内标记;以及（3）通过将该方法应用于具有高眼内压（IOP）和视神经损伤的小鼠模型的RGC。使用原代RGC培养物将允许将来仔细评估RGC中的重要细胞特异性途径，以提供对老年人群和患有视网膜神经退行性疾病的人群中视力下降的机制见解。
Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2(hi)CD48(neg)CD15(neg)CD57(neg) surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.