来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养
1School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine, 2Dongguan & Guangzhou University of Chinese Medicine Cooperative Academy of Mathematical Engineering for Chinese Medicine,Guangzhou University of Chinese Medicine, 3School of Basic Medical Sciences,Guangzhou University of Chinese Medicine
Summary
该协议试图建立一个可重复的协议，为初级神经元和胶质隔离从大鼠膀胱进一步细胞实验。
Abstract
下尿道有两个主要功能，即定期尿液储存和麦克风：这些功能通过中央和外围神经调节进行调解。虽然已经对下尿道神经系统进行了广泛的研究，但大多数研究都集中在初级文化上。该协议引入了一种从斯普拉格-道利大鼠分离和培养膀胱神经元和胶质的方法。在这种方法中，神经元和胶质在 37 °C、5% CO2 孵化器中孵育 5–7 天。因此，它们成长为适合相关后续免疫荧光实验的成熟形状。使用光学显微镜观察细胞的形态学。神经元、突触囊泡和胶质分别被β-III-图布林和MAP-2、突触-1和GFAP染色识别。同时，对几种神经递质相关蛋白质进行了免疫细胞化学，如胆碱乙酰转移酶、DYNLL2和SLC17A9。
Introduction
下尿道有两个主要功能：定期尿液储存和麦克风1。下尿道神经系统 （LUTNS） 控制这些功能，并且细腻且容易受到许多神经病变的影响， 可与生俱来（肺），获得（莱姆病），继发疾病状态（糖尿病细胞病变），药物诱导（出血性膀胱炎），手术引起的（腹部手术切除），或伤害造成的（创伤性脊髓损伤）2，3，4，5，6，7。在生理/病理学研究中，体内和体外实验同样重要。虽然在体内对LUTNS的研究已经在器官，细胞和分子水平进行了一段时间，体外研究的主要神经元从尿囊几乎不存在8，9。虽然目前的研究是有限的，但我们希望在这一领域率先进行研究，以便其他研究人员能够改进这一研究。通过这种方式，这种共同培养可能导致细胞对表型（如膀胱神经元功能障碍）中生理功能障碍的理解。
与具有明确方向性的肠道肌肉成离散层相比，膀胱的肌肉是无组织的10。因此，这种方法建议消化整个膀胱，以减少操作难度，缩短高细胞存活率的预处理时间，而不是剥落膀胱的外层。
按照这种方法，我们可以获得神经元和其他细胞的混合培养。其他细胞是必不可少的，因为它们的存在模仿了体内环境11。此外，这种细胞提供在介质中不可用的物质。
此方法涉及两个消化步骤。首先，拼贴酶II型用于水解胶原蛋白，其次是肌氨酸，将组织分离成细胞10。通过这种方式，膀胱组织被分散到单个细胞中，然后相对独立地生长。当神经元培养成熟时，神经元可用于成像或功能检测。
Protocol
所有实验协议和动物程序都符合国家研究理事会的道德原则准则。 1. 材料准备 在进行实验之前，使用高压灭菌器对所有仪器和ddH2O进行消毒。仪器包括但不限于手术剪刀、眼科剪刀、钳子、勺子核分隔器、玻璃盘（直径 60-100 mm）和玻璃破碎机。 准备Krebs解决方案如下（表1）：在使用前将所有化学物质与ddH2O一起溶解。单独溶解 CaCI…
Representative Results
在原细胞培养过程中，获得的细胞在附加状态之前具有明亮而清晰的边界。随着神经元的生长，树突和轴突开始变得与众不同。经过5-7天的培养，神经元达到一个成熟的形式与长期投影，这是理想的成像或功能研究。虽然由于介质变化，大部分杂质和细胞碎片可以清除，但可见附着在聚D-赖氨酸和层氨酸涂层上的某些残留物（图1）。 经过适当的培养，?…
Discussion
板制备
在 6、12 或 48 井培养板中使用玻璃盖片进行免疫荧光或钙成像实验是一种经济且抽样操作。在初级细胞培养的准备过程中，细胞在没有盖片的板中生长良好。因此，盖片在实验中是可有可无的，如西方斑点或聚合酶链反应。此外，涂层是电镀细胞之前的必要步骤，有或没有盖片。拉米宁和多D-lysine是涂层神经元的常见选择，尤其是层氨酸，这对神经元生长至关?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由中国国家自然科学基金委员会（第81673676号资助）和东莞市科技局（第2019622101002号资助）支持。作者感谢玛丽罗斯·沙利文博士（哈佛医学院外科助理教授）的技术咨询。
Materials
| 0.25% trypsin | Gibico | 15050065 | Enzyme digestion |
| 48-well culture plate | Corning | 3548 | Coating dish |
| antibiotic/antimycotic | Gibico | 15240062 | Culture media/Rinse media |
| Anti-Glial Fibrillary Acidic Protein Antibody | Santa Cruz | sc-33673 | ICC |
| B-27 | Gibico | 17504044 | Culture media |
| BSA Fraction V | Gibico | 332 | Enzyme digestion |
| Choline Acetyltransferase Antibody | Abcam | ab18736 | ICC |
| CO2 Incubator | Heraeus | B16UU | Cells culture |
| Collagenase type II | Sigma | 2593923 | Enzyme digestion |
| DMEM/F-12 | Gibico | 11330032 | Rinse media |
| DYNLL2 Antibody | Santa Cruz | sc-13969 | ICC |
| Fetal Bovine Serum | Gibico | 10100147 | Culture media/Rinse media |
| Forceps | Shanghai Jin Zhong Medical Devices | 1383 | 10 cm; Sterile operation |
| Glass breakers | Huan Qiu Medical Devices | 1101 | 50 ml; Sterile operation |
| Glass coverslips | WHB Scientific | WHB-48-CS | Coating dish |
| Glass dishes | Huan Qiu Medical Devices | 1177 | 100 mm; Sterile operation |
| Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488 | Southernbiotech | 3050-30 | ICC |
| Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555 | Southernbiotech | 3050-30 | ICC |
| Hoechst 33342 | BD | 561908 | ICC |
| Laminar flow bench | Su Jie Medical Devices | CB 1400V | Sterile operation |
| Laminin | Sigma | L2020 | Coating dish |
| L-glutamine | Gibico | 25030081 | Culture media |
| MAP-2 Antibody | Affinity | AF5156 | ICC |
| Murine GDNF | Peprotech | AF45044 | Culture media |
| Neurobasal-A Medium | Gibico | 10888022 | Culture media |
| Ophthalmic scissors | Shanghai Jin Zhong Medical Devices | J21010 | 12.5 cm; Sterile operation |
| Pipettes | Eppendorf | 3120000240 | 100-1000 ul; Reagent and sample pipetting |
| Pipettes | Eppendorf | 3120000267 | 10-100 ul; Reagent and sample pipetting |
| Poly-D-lysine | Sigma | P7280 | Coating dish |
| Refrigerated centrifuge | Ping Fan Instrument | TGL-16A | Enzyme digestion |
| Shaking incubator | Haimen Kylin-Bell Lab Instruments | T8-1 | Enzyme digestion |
| SLC17A9 Antibody | MBL International | BMP079 | ICC |
| Spoons nucleus divider | Shanghai Jin Zhong Medical Devices | YZR030 | 12 cm; Sterile operation |
| Substance P Antibody | Santa Cruz | sc-58591 | ICC |
| Surgical scissors | Shanghai Jin Zhong Medical Devices | J21130 | 16 cm; Sterile operation |
| Surgical towel | Fu Kang Medical Devices | 5002 | 40 x 50 cm; Sterile operation |
| Synapsin-1 Antibody | CST | 5297T | ICC |
| Tubulin beta Antibody(β-III-tubulin) | Affinity | AF7011 | ICC |
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Cite This Article
Wang, R., Huang, Z., Ren, W., Zhang, J., Zhang, Y., Tan, B., Huang, P., Cao, H. Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder. J. Vis. Exp. (159), e61177, doi:10.3791/61177 (2020).