A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.
A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.
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DOI：
10.3791/60809
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发表时间：
2020-02-14
期刊：
影响因子：
--
通讯作者：
Peters SB
中科院分区：
文献类型：
--
作者：
Barkley C;Serra R;Peters SB
牙齿神经支配使牙齿能够感知压力，温度和炎症，所有这些对牙齿器官的使用和维护至关重要。如果没有感觉神经支配，日常口腔活动将造成无法弥补的损害。尽管它的重要性，神经支配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旁分泌信号，以吸引和引导TG轴突进入和穿过牙齿。然而，很少有研究提供了详细的了解DP间充质和神经元传入之间的串扰。为了解决这一知识差距，研究人员已经开始利用共培养和各种技术来研究这些相互作用。在这里，我们展示了多个步骤参与共培养原代DP细胞与TG神经元分散在一个覆盖transwell过滤器上，大直径的孔，让轴突生长通过孔。使用腺病毒-Cre-GFP重组酶系统，利用具有侧翼为loxP位点的目的基因的原代DP细胞来促进基因缺失。使用来自Thy 1-YFP小鼠的TG神经元允许精确的传入成像，通过共聚焦显微镜观察其表达远高于背景水平。DP反应可以通过蛋白质或RNA收集和分析来研究，或者可选地，通过接种在可移除的玻璃盖玻片上的DP细胞的免疫荧光染色来研究。可以使用蛋白质组学分析等技术分析培养基，但由于培养基中存在胎牛血清，因此需要去除白蛋白。该协议提供了一个简单的方法，可以操作，以研究TG神经元和DP细胞的形态，遗传和细胞骨架的反应，在控制环境的共培养试验。
Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in coculturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.
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DOI：
10.1007/978-1-4939-7237-1_15
发表时间：
2017
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Katzenell S;Cabrera JR;North BJ;Leib DA
通讯作者：
Leib DA
DOI：
10.1016/s0169-328x(03)00335-8
发表时间：
2003-10-21
期刊：
MOLECULAR BRAIN RESEARCH
影响因子：
--
作者：
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通讯作者：
Flores, CM
影响因子：
2.7
作者：
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通讯作者：
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影响因子：
2.5
作者：
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通讯作者：
Mitrecic, Dinko
影响因子：
2.9
作者：
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通讯作者：
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