增强的绿色荧光蛋白测定，用于研究原神经元的神经质生长
Summary
在本报告中，我们描述了一个简单的方案，通过与EGFP和感兴趣的蛋白质共同转染，研究胚胎大鼠皮质神经元中的神经质外生。
Abstract
神经细胞生长是神经系统发育过程中神经回路形成的基本事件。严重的神经质损伤和突触功能障碍发生在各种神经退行性疾病和与年龄相关的退化。研究调节神经酸盐生长的机制，不仅会为大脑发育过程提供有价值的光，而且还可以揭示这些神经紊乱。由于转染效率低，目前研究特定蛋白质对原生哺乳动物神经元中神经质生长的影响具有挑战性。在这里，我们描述了一种通过与EGFP和感兴趣的蛋白质（POI）共同转染原发性大鼠皮质神经元来研究神经质外生的简单方法。该方法允许通过EGFP信号识别POI转染神经元，从而可以精确确定POI对神经质生长的影响。这种基于EGFP的测定为研究调节神经细胞生长的途径提供了一种方便的方法。
Introduction
神经酸盐，包括斧子和树突，是参与建立神经网络的神经元的投影。神经酸盐的动态生长对神经发育至关重要。然而，下面的基本监管机制仍不清楚。特别是，神经质损伤经常在各种神经退行性疾病和脑损伤后观察到1。因此，研究假定分子在各种神经酸盐生长调节途径中的作用，将增进我们对该过程的理解。此外，它可能揭示各种神经疾病的新治疗目标。神经元细胞系是研究神经元过程的宝贵模型，包括神经质生长，因为它们易于操作和转染2，3。然而，据报道，遗传漂移发生在一些常用的细胞系中，这可能导致其生理反应的变化4。此外，在神经元细胞系和主神经元之间也显示了差异蛋白表达。例如，PC12，一种来自大鼠肾上腺的神经元细胞系，广泛用于研究神经质外生长2，3，不表达NMDA受体5。此外，据提出，与原发神经元相比，小鼠神经母细胞瘤线神经-2a对神经毒素的响应能力降低是由于某些膜受体和电通道6缺乏表达。因此，原神经元是神经质生长研究更可取和更具代表性的模型。然而，主神经元的使用受到其低转染效率7的阻碍。
在这里，我们描述了一种涉及感兴趣蛋白（POI）和EGFP与原发性大鼠皮质神经元共转染的方法。EGFP作为形态标记，用于识别成功转染的神经元，并允许测量神经酸盐。我们使用已报告的化合物/分子来调节神经酸盐生长，验证了这种方法。此外，FE65，一种神经元适配器蛋白，已被证明能刺激神经质生长，用于说明这种方法8，9。该协议涉及 （1） 从胚胎第18天（E18）大鼠胚胎分离原发性皮质神经元，（2）与EGFP和POI（本研究中的FE65）共同转染神经元，以及（3）使用图像处理对神经元的成像和分析软件图像J与NeuronJ插件10，11。
Protocol
所遵循的所有程序均符合香港中文大学动物实验伦理委员会的道德标准。 1. 准备盖玻片 将无菌的 18 mm 圆形盖玻片放入 12 孔组织培养板的每个孔中。 在加湿的 37°C 培养箱中用 5 μg/mL 多 D-莱金溶液涂抹盖玻片，至少 1 小时。 从组织培养板中吸出多D-莱辛溶液，用无菌水冲洗一次涂层盖玻片。 2. 大鼠胚胎神经元解剖 在妊?…
Representative Results
为了测试这种方法，我们使用细胞D和神经生长因子NGF，这已被证明分别抑制和刺激神经质生长14，15，16。使用EGFP转染的神经元的中性粒体长度在使用Cyto D或NGF治疗后被测量。EGFP对神经元的转染效率为2.7%（计算1，068个神经元）。如图1A所示，细胞D以剂量依赖性的方?…
Discussion
如前所述，PC12及其子克隆被广泛用于研究神经酸盐延伸，因为它们具有优异的转染效率2，3。相比之下，原发神经元的转染率较低，这是通过转染7研究神经质生长调节器的主要障碍。在这里，我们描述了一个方便的协议，用于量化原始神经元中的神经质外生。尽管整体转染效率较低，但超过80%的转染神经元成功地与两种蛋白质共同转?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作由香港研究资助局、健康及医学研究基金（香港）、中大直接资助计划、联合学院捐赠基金及TUYF慈善信托基金资助。
Materials
| #5 tweezers | Regine | 5-COB | |
| 18 mm Circle Cover Slips | Thermo Scientific | CB00180RA | Sterilize before use. |
| B27 Supplement | Gibco | 17504044 | |
| Cytochalasin D | Invitrogen | PHZ1063 | Dissolved in DMSO. |
| D-(+)-Glucose | Sigma-Aldrich | G8270 | |
| Dimethyl Sulfoxide | Sigma-Aldrich | D2650 | |
| Dissecting Scissors, 10 cm | World Precision Instruments | 14393 | |
| Dissecting Scissors, 12.5 cm | World Precision Instruments | 15922 | |
| EndoFree Plasmid Maxi Kit | QIAGEN | 12362 | |
| Fluorescence Mounting Medium | Dako | S302380 | |
| Lipofectamine 2000 Transfection Reagent | Invitrogen | 11668019 | |
| Neurobasal Medium | Gibco | 21103049 | |
| NGF 2.5S Native Mouse Protein | Gibco | 13257019 | |
| Nugent Utility Forceps, 10mm, Straight Tip | World Precision Instruments | 504489 | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | |
| pEGFP-C1 | Clontech | #6084-1 | |
| pCI FE65 | Please see references 8 and 15 | ||
| PBS Tablets | Gibco | 18912014 | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Poly-D-lysine hydrobromide | Sigma-Aldrich | P7280 | |
| Spatula | Sigma-Aldrich | S4147 | |
| Trypsin-EDTA (0.05%), phenol red | Gibco | 25300062 | |
| Trypan Blue Solution, 0.4% | Gibco | 15250061 |
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Citar este artigo
Chan, W. W. R., Li, W., Chau, D. L. D., Lau, K. An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons. J. Vis. Exp. (152), e60031, doi:10.3791/60031 (2019).