研究对牙髓寄生虫信号反应的神经质生长的共同培养方法
Summary
我们描述了牙髓（DP）原细胞的分离、分散和电镀，这些细胞在覆盖的透井过滤器上培养三叉（TG）神经元。DP细胞的细胞反应可以通过免疫荧光或RNA/蛋白质分析进行分析。神经元标记物与共聚焦显微镜的免疫荧光允许分析神经质外生长反应。
Abstract
牙齿内侧使牙齿能够感知压力、温度和炎症，所有这些都对牙齿器官的使用和维护至关重要。没有感官内向，日常的口腔活动将造成无法弥补的损害。尽管它很重要，但内在牙齿在牙齿发育和维护中的作用在很大程度上被忽视了。几项研究表明，DP细胞分泌细胞外基质蛋白和副体信号，以吸引和引导TG斧头进入牙齿并在整个牙齿中。然而，很少有研究对DP间质和神经元亲和力之间的相声有详细的见解。为了解决知识的这一差距，研究人员已经开始利用共同文化和各种技术来研究这些相互作用。在这里，我们演示了在具有大直径孔隙的覆盖跨孔过滤器上分散的TG神经元共同培养初级DP细胞的多个步骤，以允许斧孔通过孔隙生长。利用腺病毒-Cre-GFP重组酶系统，利用具有致利益基因的初级DP细胞，利用loxP位点促进基因删除。使用 Thy1-YFP 鼠标中的 TG 神经元可以进行精确的对焦成像，通过共聚焦显微镜的表达远高于背景水平。DP反应可以通过蛋白质或RNA的收集和分析，或者通过免疫荧光染色的DP细胞镀在可移动玻璃盖玻片。介质可以使用技术，如蛋白组分析进行分析，虽然这将需要白蛋白耗尽，因为胎儿牛血清在介质中的存在。该协议提供了一个简单的方法，可以操纵研究TG神经元和DP细胞的形态、遗传和细胞骨骼反应，以响应共培养测定的受控环境。
Introduction
牙齿内侧使牙齿能够感知压力、温度和炎症，所有这些都对牙齿器官的使用和维护至关重要。未能感觉到与蛀牙和创伤相关的牙痛会导致疾病进展。因此，适当的内侧是正常牙齿生长、功能和护理的必要要求。
虽然大多数器官在出生时功能齐全，内在，牙齿发育延伸到成年，牙齿内侧和矿化在产后阶段1、2中协同发生。有趣的是，牙髓（DP）间质在胚胎形成过程中最初分泌排斥信号，以防止斧头进入发育中的牙齿器官，后来随着牙齿接近爆发3、4，它转移到吸引因子的分泌。在产后阶段，三角 （TG） 神经的一个发泡的斧头在牙本质沉积开始的时间渗透到牙齿中并贯穿整个牙齿（在 Pagella，P. 等人 5中进行了审查）。几项体内研究表明，神经元间质相互作用指导小鼠的牙齿内侧（在Luukko，K.等人6中回顾），但分子机制的细节很少。
细胞共培养物提供受控环境，调查人员可以在其中操纵神经元和间质体之间的相互作用。共培养实验使得深入探讨引导牙齿内向和发展的信号通路成为可能。然而，用于研究共培养细胞的几种常规方法带来了技术挑战。例如，神经酸盐生长的水晶紫罗兰染色可以非特有染色施万细胞包含在TG捆绑分散体中，并且可能存在颜色强度的峰值，反应相对较小7。微流体室提供了一个有吸引力的选择，但比跨井过滤器8，9昂贵得多，并且只允许研究神经元对DP分泌物的反应。为了解决这些问题，我们开发了一个协议，允许：a） 对TG神经质生长的精确染色和成像，以响应DP分泌物;b） 对DP细胞和/或TG神经元进行基因改造，以研究特定的信号通路，以及c）研究DP细胞对TG神经元分泌因子的反应。该协议提供了在体外共培养测定的受控环境中精确研究牙齿内侧的几个特征的能力。
Protocol
所有小鼠实验均获得UAB机构动物护理和使用委员会（IACUC）的批准。 1. 板材制备 注： 盖玻片可用于在测定结束时对 DP 细胞进行成像。在无菌组织培养罩外的所有孵育和冲皮步骤期间，确保板盖已打开，以防止样品处理过程中受到污染。 盖玻片准备 高压灭菌器圆形盖玻片。 过滤引擎盖下的超纯水。 将盖玻片?…
Representative Results
这些结果表明，与TG神经酸盐单一培养的控制相比，TG神经酸盐出生长在基础井中原DP细胞的存在下增加（图2A，C）。在神经质外生长中有一些测定到测定的变异性。因此，TG神经元单一培养应包含在所有测定中，作为检测神经质外生长基底水平的控制。在Ad-Cre-GFP和Ad-eGFP感染后，Tgfbr2f/f小鼠的初级细胞被用于此协议（图2D）。<…
Discussion
口腔的日常活动要求牙齿感知外部刺激和内部炎症，以便正确使用和维护。然而，关于驱动牙齿内侧发育过程的信号，只有有限的信息可用。该协议提供了一种分离和共同培养原DP细胞和TG神经元的方法，以研究两个群体之间的交叉通信。如下所述，对几个变量进行了优化，并留下了进一步的研究渠道。
控制在本次测定的每一步都很重要。每个测定中都应包含具有无基础DP细?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院/NIAMS（授予编号R01 AR062507和R01 AR053860至RS）的支持，b） 阿拉巴马大学伯明翰牙科学术研究培训（DART）赠款（编号T90DE022736（PI MacDougall）从国家牙科和颅面研究所/国家卫生研究院向SBP提供， c） UAB全球颅面、口腔和牙科疾病中心（GC-CODE）试点和可行性赠款给SBP和d）国家牙科和颅面研究所研究/国家卫生研究院K99 DE024406赠款给SBP。
Materials
| 5-Fluoro-2'-deoxyuridine | Sigma-Aldrich | F0503 | Used as a mitotic Inhibitor at 15 μM concentration in co-culture media, Day 2 |
| 24 Well Cell Culture Plate | Corning | 3524 | Co-culture plate |
| Alexa-546 anti-chicken | Invitrogen | A-11040 | Secondary to stain neurite outgrowth labeled by anti-GFP antibody, 1:500 dilution |
| Anti-GFP Antibody | Aves Lab, Inc | GFP-1010 | Primary antibody to label Thy1-YFP neurons, 1:200 dilution |
| Anti-Neurofilament 200 antibody | Sigma-Aldrich | NO142 | Monoclonal primary antibody to label neurons, 1:1000 dilution, alternative if YFP mice are not available |
| B6;129- Tgfbr2tm1Karl/J | The Jackson Laboratory | 12603 | Tgfbr2f/f mouse model used for dental pulp cells in optimized protocol |
| B6.Cg-Tg(Thy1-YFP)16Jrs/J | The Jackson Laboratory | 3709 | Thy1-YFP mouse model genotype used for trigeminal neurons |
| Collagenase Type II | Millipore | 234155-100MG | Used to disperse trigeminal neurons |
| Fetal Bovine Serum | Gibco | 10437 | Additive to co-culture media |
| Fine forceps | Fine Science Tools | 11413-11 | Fine forceps for TG dissection |
| Laminin | Sigma-Aldrich | L2020 | Coats the transwell inserts at final concentration of 10 μg/ml, stock solution is assumed at 1.5 mg/ml |
| Lysis Buffer (Buffer RLT) | Qiagen | 79216 | Extracts RNA from dental pulp cells post co-culture |
| L-Glutamine | Gibco | 25030081 | Additive to co-culture media |
| Micro-dissecting scissors | Sigma-Aldrich | S3146-1EA | Dissection scissors to open skull |
| Microscope Cover Glass | Fisherbrand | 12-545-81 | Circlular coverslip for optional cell culturing and immunofluorescence processing |
| Minimal Essential Medium a | Gibco | 12571063 | Co-culture media base |
| Penicillin-Streptomycin | Gibco | 15070063 | Antibiotic additive to co-culture media |
| Phosphatase Inhibitor | Sigma-Aldrich | 04 906 837 001 | Additive to RIPA Buffer for extracting protein from dental pulp cells post co-culture |
| Polybrene | Millipore | TR-1003-G | Used to aid in dental pulp cell transfection |
| Poly-D-Lysine | Sigma-Aldrich | P7280 | Coverslip coating to aid dental pulp cellular adhesion |
| Protease Inhibitors | Millipore | 05 892 791 001 | Additive to RIPA Buffer for extracting protein from dental pulp cells post co-culture |
| RNAse/DNAse free eppendorf tubes | Denville | C-2172 | Presterilized 1.7 ml tubes for RNA, DNA or protein collection at the end of assay |
| ThinCert Cell Culture Insert | Greiner Bio-One | 662631 | Transwell inserts for trigeminal neurons in co-culture assays |
| Trypsin-EDTA (0.25%) | Gibco | 25200056 | Used fto disperse dental pulp cells |
| Trypsin Type II | Sigma-Aldrich | T-7409 | Used to disperse trigeminal neurons |
| Ultra Fine Forceps | Fine Science Tools | 11370-40 | Ultra fine forceps for dissection |
| Uridine | Sigma-Aldrich | U3750 | Used as a mitotic Inhibitor at 1 μM concentration in co-culture media, Day 2 |
| Vacuum Filtration System | Millipore | SCNY00060 | Steriflip disposable filter, 50 μm nylon net filter |
| Vial forceps | Fine Science Tools | 110006-15 | Long forceps for tissue transfer to conicals |
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Diesen Artikel zitieren
Barkley, C., Serra, R., Peters, S. B. A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals. J. Vis. Exp. (156), e60809, doi:10.3791/60809 (2020).