Name: purification of fibroblasts and schwann cells from sensory and motor nerves in vitro
Uploaded: 2020-05-20T13:00:00
Description: Watch this Scientific Journal Video about Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro at JoVE.com

这项研究得到了中国国家重点研究发展计划（赠款2017YFA0104703），中国国家自然基金会（赠款号31500927）的支持。

本研究是根据南通大学《机构动物保护指南》进行的。包括动物主体在内的所有程序都经过中国江苏省实验动物管理委员会的道德批准。
1. 运动和感觉神经成纤维细胞和SC的隔离与培养
<ol><li>使用中国南通大学实验动物中心提供的七天大的斯普拉格-道利（SD）大鼠（n=4）。将大鼠放在含有5%异氟素的水箱中2-3分钟，让动物缓慢呼吸，没有独立活动，然后在斩首前使用75%乙醇?...

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周围神经的两个主要细胞群包括SC和成纤维细胞。主要培养的成纤维细胞和SC可以准确地帮助在外周神经发育和再生期间对成纤维细胞和SC的生理学进行建模。研究表明，P7大鼠坐骨神经细胞含有约85%的S100阳性SCs，13%的OX7阳性成纤维细胞和只有1.5%的OX42阳性巨噬细胞13。虽然成纤维细胞的数量小于SCs，但成纤维细胞的初始增殖率比SCs快。因此，Ara-C是许多研究中最常用的抗米蒂剂，?...

在外周神经系统中，神经纤维主要由轴子、施万细胞（SCs）和成纤维细胞组成，还含有少量的巨噬细胞、微血管内皮细胞和免疫细胞1。SCs 以 1：1 的比例包裹轴子，并由称为内结组织层的连接组织层封闭。轴子然后捆绑在一起形成称为分柱的组，每个分膜被包裹在称为"内皮"的结缔组织层中。最后，整个神经纤维被包裹在一层结缔组织中，称为肾上腺素。在末体中，整个细胞群体由48%的SC组成，其余细胞中相当一部分涉及成纤维细胞2。此外，成纤维细胞是所有神经隔间的重要组成部分，包括肾上腺素、内膜和内膜3。许多研究表明，在外周神经损伤4、5、6之后，SCs和成纤维细胞4在再生过程中起着至关重要的作用。外周神经转切后，周利尿成纤维细胞通过SCs和成纤维细胞之间的ephrin-B/EphB2信号通路调节细胞分选，进一步引导弓弦再生通过伤口5。外周神经成纤维细胞分泌泰纳辛-C蛋白，并通过+1-integrin信号通路7增强SCs在神经再生期间的迁移。然而，上述研究中使用的SCs和成纤维细胞来自坐骨神经，包括感觉神经和运动神经。
在外周神经系统中，感觉神经（发热神经）从受体到中枢神经系统（CNS）进行感官信号，而运动神经（发热神经）则从CNS传导到肌肉的信号。先前的研究表明，SCs表达不同的运动和感官表型和分泌神经营养因子，以支持周围神经再生,8，9。根据最近的一项研究，成纤维细胞也表达不同的运动和感官表型，并影响SC10的迁移。因此，探索运动和感觉神经成纤维细胞/SCs之间的差异使我们能够研究周围神经特异性再生的复杂底层分子机制。
目前，有许多方法可以净化SCs和成纤维细胞，包括应用抗米氏剂、抗体中联细胞分析11、12、,12顺序免疫平移13和层压基14。然而，上述所有方法都去除成纤维细胞，只保留SCs。高度纯化的SC和成纤维细胞可以通过流式细胞分理技术获得，但它是一种耗时且昂贵的技术。因此，在这项研究中，开发了一种简单的差分消化和差分粘附方法，用于净化和隔离感觉和运动神经成纤维细胞和SC，以便更快获得大量的成纤维细胞和SC。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 594 Goat Anti-Mouse IgG(H+L)</td>
			<td>Life Technologies</td>
			<td>A11005</td>
			<td>Dilution: 1:400</td>
		</tr>
		<tr>
			<td>CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG(H+L)</td>
			<td>Proteintech</td>
			<td>SA00013-1</td>
			<td>Dilution: 1:400</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Quest software</td>
			<td>Becton Dickinson Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-Hank&#39;s balanced salt solution</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus</td>
			<td>SZ2-ILST</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10099-141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield Mounting Medium</td>
			<td>Abcam</td>
			<td>ab104135</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation medium/Permeabilization medium</td>
			<td>Multi Sciences (LIANKE) Biotech, Co., LTD</td>
			<td>GAS005</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry</td>
			<td>Becton Dickinson Biosciences</td>
			<td>FACS Calibur</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 kappa [MOPC-21] (FITC) - Isotype Control</td>
			<td>Abcam</td>
			<td>ab106163</td>
			<td>Dilution: 1:400</td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-CD90 antibody</td>
			<td>Abcam</td>
			<td>ab225</td>
			<td>Dilution: 1:1000 for ICC, 0.1 &#181;g for 106 cells for Flow Cyt</td>
		</tr>
		<tr>
			<td>Mouse anti-S100 antibody</td>
			<td>Abcam</td>
			<td>ab212816</td>
			<td>Dilution: 1:400</td>
		</tr>
		<tr>
			<td>Polylysine (PLL)</td>
			<td>Sigma</td>
			<td>P4832</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human NRG1-beta 1/HRG1-beta 1 EGF Domain Protein</td>
			<td>R&#38;D Systems</td>
			<td>396-HB-050</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% (w/v) trypsin</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of fibroblasts and schwann cells from sensory and motor nerves in vitro

周围神经系统的主要细胞是施万细胞（SCs）和成纤维细胞。这两个细胞都清楚地表达在神经营养因子基因表达和其他生物过程的不同模式中涉及的感官和运动表型，影响神经再生。本研究已建立一种方案，以更快获得高度纯化的老鼠感官和运动SCS和成纤维细胞。新生儿大鼠的腹根（运动神经）和后根（感觉神经）分离，细胞培养4-5天，随后通过组合差分消化和差分粘附方法分离感觉和运动成纤维细胞和SC。免疫细胞化学和流式细胞学分析结果表明，感觉和运动SCs和成纤维细胞的纯度为>90%。该协议可用于获得大量的感官和运动成纤维细胞/SCs更快，有助于探索感官和运动神经再生。

光显微观察 SCs和成纤维细胞是从神经组织中获得的原细胞培养中的两个主要细胞群。接种1小时后，大部分细胞粘附在培养皿底部，细胞形态由圆形改为椭圆形。经过24小时培养后，SCs表现出双极性或三极形态，其长度从100微米到200μm之间。48小时后，细胞发生聚集和增殖，其中许多细胞被聚合在端到端，肩对肩，漩涡或围栏一样排列。其他比SCS大的成纤维?...

Watch this Scientific Journal Video about Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro at JoVE.com

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

在这里，我们提出了一种方法，从体外感觉和运动神经中纯化成纤维细胞和Schwann细胞。

从体外感觉和运动神经中纯化成纤维细胞和施万细胞

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

该协议可用于快速获取大量的传感器和电机成纤维细胞和施万细胞。通过此信息获得的细胞可用于神经再生和神经系统疾病的研究。我们演示可以代表实验，更好地控制微分消化步骤的时间，使实验步骤更容易理解。要用剪刀爆裂，做一个三厘米的切口，在背部的皮肤。拆下脊柱。小心地打开椎管，露出脊髓。将脊髓系在60毫米培养皿中，在解剖显微镜下用三到四毫升的冰冷D汉克斯平衡盐溶液，切除腹根和背根。将它们放在冰冷的D汉克斯平衡盐溶液中。取出HSS后，用剪刀将神经切成三到五毫米。加入一毫升0.25%的三辛，并将神经片段转移到5毫升离心管。在37摄氏度下孵育18至20分钟。接下来，为了停止消化，在含有10%胎儿牛血清的三到四毫升DMEM中加入。轻轻上下移液混合物约10次。在 800 次 G 下离心五分钟。离心后丢弃上经剂。并在两到三毫升DMEM中重新悬浮沉淀物，并辅以10%FBS。通过 400 网状过滤器过滤细胞悬浮液，然后使用悬浮液为 60 毫米 Petri 盘接种。在37摄氏度下孵育培养培养皿4至5天，同时存在5%的二氧化碳。当培养达到90%汇合时，使用1XPBS清洗细胞一次。然后，要消化Schwann细胞，每60毫米培养皿在37摄氏度下加入1毫升0.25%的三丁鱼。在室温下8至10秒后，停止消化，加入3毫升DMEM，辅以10%FBS。要分离Schwann细胞，用移液器轻轻吹在细胞上。用显微镜验证细胞是否分离。然后收集含有天鹅细胞的介质，并在800次G下离心5分钟。丢弃上一杯，并在三毫升的介质中重新悬浮沉淀物。使用此悬浮液为未涂覆的 60 毫米培养皿接种，并在 37 摄氏度下孵育盘子 30 至 45 分钟。该培养基的品牌，其中将施万细胞到聚L赖氨酸涂层的培养皿，并在37摄氏度的温度下孵育两天。去除含有Schwann细胞的培养基后，用1XPBS粘附在培养皿上。要消化成纤维细胞，在37摄氏度下加入0.25%的三丁辛1毫升。消化后，在37摄氏度下进行两分钟，通过添加DMEM和10%FBS来结束消化。要分离成纤维细胞，请使用移液器吹在盘子的底部。收集介质，包括成纤维细胞，并转移到离心管。在800次G下离心管5分钟。丢弃上一杯，用含有10%FBS的两毫升DMEM重新沉淀物。在未编码的60毫米、培养皿中接种细胞，并在37摄氏度下孵育30至45分钟。通过去除和丢弃生长介质，分离将粘附在培养皿中的成纤维细胞。加入三毫升DMEM，辅以10%FBS。在37摄氏度下孵育两天，直到通道一个细胞达到90%的汇合。重复差分消化和粘附。在培养成纤维细胞和施万细胞两天后，消化细胞，收集细胞，并计算它们。接种PLL涂层幻灯片的浓度是每井10个细胞的浓度，用于ICC染色。Bay 的对比显微镜显示了在整个分离过程中培养的施万细胞和成纤维细胞的形态。经过长时间的培养，施万细胞聚集在成纤维细胞之间或位于成纤维细胞表面。消化10秒后，以红色箭头表示的Schwann细胞是圆的，而成纤维细胞保持平坦，并附着在盘子的底部。通过差分消化和差分粘附分离后，所有四种细胞类型都观察到典型的细胞形态。运动成纤维细胞，感觉成纤维细胞，运动施万细胞和感觉施万细胞。使用锥形焦激光扫描显微镜对感觉和运动成纤维细胞进行可视化。成纤维细胞被染色的抗体对CD 90 X33342染料被用来标记细胞核。合并的成纤维细胞免疫染色和核染色图像显示，在CD90和六边原共标记细胞中，分别有92.51%和92.64%存在于运动细胞和感觉成纤维细胞中。使用共声激光扫描显微镜对感觉和运动施万细胞进行可视化。Schwann细胞被染色的抗体对S100 X33342染料被用来标记细胞核。天鹅细胞免疫染色和核染色的合并图像表明，S100和六边原共标记细胞分别存在91.61%和93.56%。还使用流式细胞学对细胞纯度进行了分析。在成纤维细胞培养中，大约90%的细胞是成纤维细胞，在FCA图中由M2表示，而其余细胞是施万细胞。在Schwann细胞培养中，超过92%的细胞是Schwann细胞，在FCA图中，M2再次表明，而其余细胞是成纤维细胞。该协议很少可用于启动成纤维细胞的传感器和运动神经再生机制，以及Schwann细胞移植，以促进神经再生。

Research

JoVE Journal

Neuroscience

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

初级运动和感觉神经元上的静电聚- L -乳酸纳米纤维支架中定义的媒体文化

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

科研

神经科学

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

去除<em&gt;果蝇</em从幼虫鱼片的感官神经元和表皮细胞的免疫荧光分析&gt;肌肉组织

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

从美国牛蛙的小囊（毛细胞的生理准备<em&gt;牛蛙</em&gt;）

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

体外刺激对龟脑神经的运动测量

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...