一步冷常压等离子体体外处理神经干细胞分化
Summary
该方案旨在提供神经干细胞冷大气等离子体处理和免疫荧光检测的详细实验步骤, 以提高分化。
Abstract
随着物理等离子体技术的发展, 冷大气等离子体 (cap) 在去污、癌症治疗、创面愈合、根管治疗等方面得到了广泛的研究, 形成了一个新的研究领域–血浆医学。作为电、化学和生物反应物种的混合物, cap 已经证明了它们在体外和体内增强神经干细胞分化的能力, 正在成为神经疾病治疗的一种有希望的方法在未来。更令人兴奋的消息是, 使用 cap 可以实现一步, 并安全定向, 分化神经干细胞 (nsc) 的组织运输。本文演示了利用自制 cap 射流装置增强 c17.2-nsc 和原发性大鼠神经干细胞 nsc 分化的详细实验方案, 以及倒置显微镜和荧光显微镜观察细胞命运的实验方案。利用免疫荧光染色技术, 发现 nsc 均比未治疗组表现出加速差异率, ~ 75% 的 nsc 选择性分化为神经元, 主要是成熟的、胆碱能的和运动的。神经元。
Introduction
nsc 定向分化为一定的组织转运谱系被认为是治疗神经退行性疾病和神经创伤性疾病最有前途的方法之一。例如, 儿茶酚胺能多巴胺能神经元是帕金森病 (pd) 治疗中特别需要的。然而, 传统的方法, 准备所需的细胞运输有许多缺点, 如化学毒性, 疤痕形成, 或其他, 这在很大程度上阻碍了 nsc 在再生医学2的应用。因此, 寻找一种新颖、安全的 nsc 分化方法是非常必要的。
等离子体是继固体、液体和气体之后的第四种物质状态, 它占整个宇宙物质的95% 以上。等离子体与未结合的正向粒子/负粒子和中性粒子呈电中性, 通常由实验室中的高压放电产生。近二十年来, 随着冷大气压力等离子体技术的发展, 等离子体在生物医学中的应用受到了广泛的关注。通过这项技术, 可以在大气中的大气中产生稳定的低温等离子体, 而不会形成电弧, 它由各种活性物质组成, 如活性氮种 (rns)、活性氧物种 (ros)、紫外线 (uv)辐射、电子、离子和电场3。cap 在微生物失活、癌症治疗、伤口愈合、皮肤病治疗、细胞增殖和细胞分化4、5、6、7等方面具有独特的优势。在前面的研究中, 我们证明了冷大气等离子体射流可以增强 nsc 在小鼠神经干细胞 c17.2 (c17.2-nsc) 和原代大鼠神经干细胞中的分化, 显示出成为定向靶向的强大工具的巨大潜力。nsc8的分化。虽然目前还没有完全了解 nsc 分化的 cap 增强机制, 但 cap 产生的 no 已被证明是这一过程中的关键因素。在这项工作中, 我们的目的是提供一个详细的实验方案, 使用大气压力螺旋氧等离子体喷射器治疗 nsc 的体外, 细胞的制备和预处理, 形态学观察倒置显微镜,荧光显微镜观察免疫荧光染色。
Protocol
Representative Results
Discussion
c17.2-nsc 是由 snyder 等人开发的一种由新生小鼠小脑颗粒层细胞产生的不朽神经干细胞.c17.2-nsc 可分化为神经元、星形胶质细胞和少突胶质细胞, 广泛应用于神经科学12。在我们之前的研究中, cap 可以增强 c17.2-nsc 向神经元的分化。还使用原发性大鼠 nsc 进行了原理验证研究, 血浆暴露对原发性大鼠 nsc 的影响在质量上与 c17.2 nsc 相似, 神经?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了华中学者计划、华中科技大学自主创新基金 (2018kfyxj071 号) 和中国国家自然科学基金 (31501099 和 51707012) 的支持。
Materials
| Coverslip | NEST | 801008 | |
| Poly-D-lysine | Beyotime | P0128 | |
| DMEM medium | HyClone | SH30022.01B | stored at 4 °C |
| DMEM/F12 medium | HyClone | SH30023.01B | stored at 4 °C |
| N2 supplement | Gibco | 17502048 | stored at -20 °C and protect from light |
| B27 supplement | Gibco | 17504044 | stored at -20 °C and protect from light |
| Fetal bovine serum | HyClone | SH30084.03 | stored at -20 °C, avoid repeated freezing and thawing |
| Donor Horse serum | HyClone | SH30074.03 | tored at -20 °C, avoid repeated freezing and thawing |
| Penicillin/Streptomycin | HyClone | SV30010 | stored at 4 °C |
| Trypsin | HyClone | 25300054 | stored at 4 °C |
| PBS solution | HyClone | SH30256.01B | stored at 4 °C |
| 4% paraformaldehyde | Beyotime | P0098 | stored at -20 °C |
| TritonX-100 | Sigma | T8787 | |
| Normal Goat Serum Blocking Solution | Vector Laboratories | S-1000-20 | stored at 4 °C |
| anti-Nestin | Beyotime | AF2215 | stored at -20 °C, avoid repeated freezing and thawing |
| anti-β-Tubulin III | Sigma Aldrich | T2200 | stored at -20 °C, avoid repeated freezing and thawing |
| anti-O4 | R&D Systems | MAB1326 | stored at -20 °C, avoid repeated freezing and thawing |
| anti-NF200 | Sigma | stored at -20 °C, avoid repeated freezing and thawing | |
| anti-ChAT | Sigma | stored at -20 °C, avoid repeated freezing and thawing | |
| anti- LHX3 | Sigma | stored at -20 °C, avoid repeated freezing and thawing | |
| anti-GABA | Sigma | stored at -20 °C, avoid repeated freezing and thawing | |
| anti-Serotonin | Abcam, Cambridge, MA | stored at -20 °C, avoid repeated freezing and thawing | |
| anti-TH | Abcam, Cambridge, MA | stored at -20 °C, avoid repeated freezing and thawing | |
| Immunol Staining Primary Antibody Dilution Buffer | Beyotime | P0103 | stored at 4 °C |
| Cy3 Labeled Goat Anti-Rabbit IgG | Beyotime | A0516 | stored at -20 °C and protect from light |
| Alexa Fluor 488- Labeled Goat | Beyotime | A0428 | stored at -20 °C and protect from light |
| Anti-Mouse IgG | |||
| 12-well plate | corning | 3512 | |
| 25 cm2 flask | corning | 430639 | |
| Hoechst 33258 | Beyotime | C1018 | stored at -20 °C and protect from light |
| Mounting medium | Beyotime | P0128 | stored at -20 °C and protect from light |
| Light microscope | Nanjing Jiangnan Novel Optics Company | XD-202 | |
| Fluorescence microscopy | Nikon | 80i | |
| High – voltage Power Amplifier | Directed Energy | PVX-4110 | |
| DC power supply | Spellman | SL1200 | |
| Function Generator | Aligent | 33521A | |
| Oscilloscope | Tektronix | DPO3034 | |
| High voltage probe | Tektronix | P6015A |
Referências
- Temple, S. The development of neural stem cells. Nature. 414 (6859), 112-117 (2001).
- Rossi, F., Cattaneo, E. Neural stem cell therapy for neurological diseases: dreams and reality. Nature Reviews Neuroscience. 3 (5), 401-409 (2002).
- Graves, D. B. The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology. Journal of Physics D: Applied Physics. 45 (26), 263001 (2012).
- Weltmann, K. D., von Woedtke, T. Plasma medicine-current state of research and medical application. Plasma Physics and Controlled Fusion. 59 (1), 014031 (2017).
- Lloyd, G., et al. Gas Plasma: Medical Uses and Developments in Wound Care. Plasma Processes and Polymers. 7 (3-4), 194-211 (2010).
- Yousfi, M., Merbahi, N., Pathak, A., Eichwald, O. Low-temperature plasmas at atmospheric pressure: toward new pharmaceutical treatments in medicine. Fundamental & Clinical Pharmacology. 28 (2), 123-135 (2014).
- Xiong, Z., Roe, J., Grammer, T. C., Graves, D. B. Plasma Treatment of Onychomycosis. Plasma Processes and Polymers. 13 (6), 588-597 (2016).
- Xiong, Z., et al. Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation. Stem Cell Research. 12 (2), 387-399 (2014).
- Xie, Z., Zheng, Q., Guo, X., Yi, C., Wu, Y. Isolation, Culture and Identification of Neural Stem Cells in New-born Rats. Journal of Huazhong University of Science and Technology. [Med Sci]. 23 (2), 75-78 (2003).
- Ryder, E. F., Snyder, E. Y., Cepko, C. L. Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer. Journal of Neurobiology. 21 (2), 356-375 (1990).
- Snyder, E. Y., Taylor, R. M., Wolfe, J. H. Neural progenitor cell engraftment corrects lysosomal storage throughout the MRS VII mouse brain. Nature. 374 (6520), 367-370 (1995).
- Snyder, E. Y., Yoon, C., Flax, J. D., Macklis, J. D. Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proceedings of the National Academy of Sciences of the United States of America. 94 (21), 11663-11668 (1997).
- Jamur, M. C., Oliver, C. Cell Fixatives for Immunostaining. Methods in Molecular Biology. 588, 55-61 (2010).
- Jamur, M. C., Oliver, C. Permeabilization of Cell Membranes. Methods in Molecular Biology. 588, 63-66 (2010).
