脂肪来源的间充质基质细胞与原代混合胶质细胞共培养，以减少朊病毒诱导的炎症
1Prion Research Center,Colorado State University, 2Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences,Colorado State University, 3Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences,Colorado State University
Summary
脂肪来源的间充质基质细胞 （AdMSC） 具有有效的免疫调节特性，可用于治疗与炎症相关的疾病。我们演示了如何分离和培养小鼠 AdMSCs 和原代混合胶质细胞，刺激 AdMSCs 上调抗炎基因和生长因子，评估 AdMSCs 的迁移，以及将 AdMSCs 与原代混合朊病毒感染的神经胶质细胞共培养。
Abstract
间充质基质细胞 （MSC） 是通过产生抗炎细胞因子、趋化因子和生长因子来有效调节炎症的。这些细胞在神经退行性疾病（如朊病毒病和其他蛋白质错误折叠疾病）的背景下显示出调节神经炎症的能力。朊病毒病可以是散发的、获得性的或遗传性的;它们可能是由于朊病毒蛋白在大脑中的错误折叠和聚集造成的。这些疾病总是致命的，没有可用的治疗方法。
疾病的最早迹象之一是星形胶质细胞和小胶质细胞的激活以及相关的炎症，这发生在可检测到的朊病毒聚集和神经元丢失之前;因此，MSCs的抗炎和调节特性可用于治疗朊病毒病中的星形胶质细胞增生。最近，我们发现脂肪来源的间充质干细胞（AdMSCs）与BV2细胞或原代混合胶质细胞共培养，通过旁分泌信号传导减少朊病毒诱导的炎症。本文描述了一种使用刺激的AdMSCs来减少朊病毒诱导的炎症的可靠治疗方法。
AdMSCs的杂合子群体可以很容易地从小鼠脂肪组织中分离出来，并在培养物中扩增。用炎性细胞因子刺激这些细胞可增强它们向朊病毒感染的大脑匀浆迁移并产生抗炎调节剂作为反应的能力。总之，这些技术可用于研究间充质干细胞对朊病毒感染的治疗潜力，并可用于其他蛋白质错误折叠和神经炎症性疾病。
Introduction
神经胶质炎症在多种神经退行性疾病中起着关键作用，包括帕金森氏症、阿尔茨海默氏症和朊病毒病。尽管异常蛋白质聚集归因于大部分疾病发病机制和神经退行性变，但神经胶质细胞也在加剧这种 1,2,3 中起作用。因此，靶向神经胶质诱导的炎症是一种很有前途的治疗方法。在朊病毒病中，细胞朊病毒蛋白 （PrPC） 与疾病相关的朊病毒蛋白 （PrPSc） 错误折叠，后者形成寡聚体和聚集体并破坏大脑中的稳态 4,5,6。
朊病毒病的最早迹象之一是星形胶质细胞和小胶质细胞的炎症反应。通过去除小胶质细胞或修饰星形胶质细胞来抑制这种反应的研究通常显示，在动物模型中，疾病发病机制没有改善或恶化 7,8,9。在不消除胶质炎症的情况下调节神经胶质炎症是一种有趣的治疗方法。
间充质基质细胞 （MSCs） 因其能够以旁分泌方式调节炎症而成为治疗多种炎症性疾病的舞台 10,11。它们已经显示出迁移到炎症部位的能力，并通过分泌抗炎分子、生长因子、microRNA 等来对这些环境中的信号分子做出反应 10,12,13。我们之前已经证明，源自脂肪组织的间充质干细胞（表示为 AdMSCs）能够迁移到朊病毒感染的脑匀浆体，并通过上调抗炎细胞因子和生长因子的基因表达来对这种脑匀浆做出反应。
此外，AdMSCs 可以降低 BV2 小胶质细胞和原代混合胶质细胞 14 中与核因子-κB （NF-κB）、含 3 的 Nod 样受体家族 pyrin 结构域 （NLRP3） 炎症小体信号传导和神经胶质细胞激活相关的基因表达。在这里，我们提供了如何从小鼠中分离AdMSCs和原代混合胶质细胞，刺激AdMSCs上调调节基因，评估AdMSCs迁移以及将AdMSCs与朊病毒感染的胶质细胞共培养的方案。我们希望这些程序可以为进一步研究MSCs在调节神经退行性疾病和其他疾病中神经胶质诱导的炎症中的作用提供基础。
Protocol
小鼠在科罗拉多州立大学的实验室动物资源中心繁殖和维护，该资源公司由实验室动物护理国际评估和认证协会认证，符合协议#1138，由科罗拉多州立大学机构动物护理和使用委员会批准。 1.用朊病毒分离和感染原代皮质混合胶质细胞 为了分离含有星形胶质细胞和小胶质细胞的初级混合胶质细胞，获得0至2天大的C57Bl / 6小鼠幼崽。注意：该协议改编自以…
Representative Results
用TNFα或干扰素-γ（IFNγ）刺激AdMSCs24小时诱导抗炎分子和生长因子表达的变化。用 TNFα 或干扰素-γ （IFNγ） 处理 AdMSCs 会增加 TNF 刺激的基因 6 （TSG-6） mRNA，而 TNFα 而不是 IFNγ 会导致转化生长因子 β-1 （TGFβ-1） mRNA 的增加。用TNFα或IFNγ刺激可诱导血管内皮生长因子（VEGF）mRNA增加，但成纤维细胞生长因子（FGF）mRNA的表达没有变化（图1A）。这些数据表明，AdM…
Discussion
在这里，我们展示了一种可靠且相对便宜的方案，用于评估脂肪来源的间充质基质细胞（AdMSCs）在神经胶质细胞模型中减少朊病毒诱导的炎症的影响。AdMSCs可以很容易地在培养物中分离和扩增，可在短短1周内使用。该方案一致地产生异源细胞群，这些细胞群通过免疫荧光和流式细胞术表达与间充质基质细胞一致的标记物，并且在引入细胞因子或朊病毒感染的脑匀浆时保持免疫功能14<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Lab Animal Resources的畜牧业。我们撰写这篇手稿的资金来源包括 Boettcher 基金、Murphy Turner 基金、科罗拉多州立大学兽医学院和生物医学科学学院研究委员会。 图2A、图2C 和 图3A 是用 BioRender.com 创建的。
Materials
| 0.25% Trypsin | Cytiva | SH30042.01 | |
| 5 mL serological pipets | Celltreat | 229005B | |
| 6-well tissue culture plates | Celltreat | 229106 | |
| 10 cm cell culture dishes | Peak Serum | PS-4002 | |
| 10 ml serological pipets | Celltreat | 229210 | |
| 15 mL conical tubes | Celltreat | 667015B | |
| 50 mL conical tubes | Celltreat | 667050B | |
| BV2 microglia cell line | AcceGen Biotech | ABC-TC212S | |
| Cell lifter | Biologix Research Company | 70-2180 | |
| Crystal violet | Electron Microscopy Sciences | 12785 | |
| Dispase | Thermo Scientific | 17105041 | |
| DMEM/F12 | Caisson Labs | DFL14-500ML | |
| DNase-I | Sigma Aldrich | 11284932001 | |
| Essential amino acids | Thermo Scientific | 11130051 | |
| Ethanol (100%) | EMD Millipore | EX0276-1 | |
| Fetal bovine serum (heat inactivated) | Peak Serum | PS-FB4 | Can be purchased as heat inactivated or inactivated in the laboratory |
| Formaldehyde | EMD Millipore | 1.04003.1000 | |
| Glass 10 mL serological pipet | Corning | 7077-10N | |
| Hank’s Balances Salt Solution | Sigma Aldrich | H8264-500ML | |
| Hemocytometer/Neubauer Chamber | Daigger | HU-3100 | |
| High Glucose DMEM | Cytiva | SH30022.01 | |
| low glucose DMEM containing L-glutamine | Cytiva | SH30021.01 | |
| MEM/EBSS | Cytiva | SH30024.FS | |
| non-essential amino acids | Sigma-Aldrich | M7145-100M | |
| Paraformaldehyde (16%) | MP Biomedicals | 219998320 | |
| Penicillin/streptomycin/neomycin | Sigma-Aldrich | P4083-100ML | |
| Phosphate buffered saline | Cytiva | SH30256.01 | |
| Recombinant Mouse IFN-gamma Protein | R&D Systems | 485-MI | |
| Recombinant Mouse TNF-alpha (aa 80-235) Protein, CF | R&D Systems | 410-MT | |
| RNeasy mini kit | Qiagen | 74104 | |
| Sigmacote | Sigma Aldrich | SL2-100ML | Coat inside of glass pipets by aspirating up and down twice in Sigmacote and allowing to dry thoroughly. Wrap in aluminum foil and autoclave pipets 24 h later. |
| Stemxyme | Worthington Biochemical Corporation | LS004106 | Collagenase/Dispase mixture |
| Sterile, individually wrapped cotton swab | Puritan Medical | 25-8061WC | |
| Thincert Tissue Culture Inserts, 24 well, Pore Size=8 µm | Greiner Bio-One | 662638 | |
| Thincert Tissue Culture Inserts, 6 well, Pore Size=0.4 µm | Greiner Bio-One | 657641 |
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Cite This Article
Hay, A. J. D., Popichak, K. A., Zabel, M. D., Moreno, J. A. Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation. J. Vis. Exp. (198), e65565, doi:10.3791/65565 (2023).