Name: microtubule plus-end dynamics visualization in huntington's disease model based on human primary skin fibroblasts
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Description: Watch this Scientific Journal Video about Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts at JoVE.com

该研究由俄罗斯联邦科学和高等教育部资助，拨款号为075-15-2019-1669（成纤维细胞转染），由俄罗斯科学基金会资助，拨款号为19-15-00425（所有其他关于 体外培养成纤维细胞的工作）。它得到了莫斯科国立罗蒙诺索夫大学发展计划PNR5.13（成像和分析）的部分支持。作者感谢尼康卓越中心在A. N. Belozersky物理化学生物学研究所的支持。我们要特别感谢叶卡捷琳娜·塔兰（Ekaterina Taran）在配音方面的帮助。作者还感谢Pavel Belikov在视频编辑方面的帮助。手稿中的人物是用 BioRender.com 创作的。

该协议遵循2015年9月8日联邦医学生物局物理化学医学研究和临床中心的指南。
注： 图 1 概述了该协议。
1. 获得人皮肤成纤维细胞的初级培养物（图2）
<ol><li>在Dulbecco的改良鹰培养基（DMEM）培养基中，补充50μg/ mL青霉素和50 U / mL链霉素，在几个小时内将活检送至实验室。 注意：在...

<ol>
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从高质量的显微图像中可以获得更高质量的微管动力学分析结果。重要的是要观察活细胞延时成像的所有必要条件，并正确调整成像参数。使用带有玻璃底的特殊细胞培养皿（共聚焦培养皿）很重要，因为玻璃具有与塑料不同的光折射率。玻璃的厚度及其在整个区域内的均匀性也非常重要，因为这些参数对于正常的样品聚焦至关重要。违反这些参数不可避免地会导致完美对焦系统故障，从而导致?...

亨廷顿舞蹈症（HD）是一种无法治愈的神经退行性病理学，由编码亨廷顿蛋白（HTT）的基因突变引起。HTT主要与囊泡和微管有关，可能参与微管依赖性转运过程1，2。为了研究突变HTT对微管动力学的影响，我们使用EB3蛋白的体外可视化，通过结合和稳定生长的正端来调节微管的动态特性。为了将荧光标记的EB3加载到人皮肤成纤维细胞中，应用质粒转染。在本研究中，我们使用从HD患者皮肤活检中获得的原发性成纤维细胞培养物。
HTT蛋白基因的突变导致聚谷氨酰胺束3的伸长。HTT在细胞内吞作用4、细胞转运1、2、蛋白质降解5等细胞过程中起作用。这些过程的很大一部分涉及细胞骨架的各种元件，包括微管。
人类原代细胞是尽可能密切地再现患者细胞中发生的事件的最佳模型。要创建这样的模型，需要从人体活检材料（例如，从手术样本中）分离细胞。所得原代细胞系适合于使用各种遗传、生化、分子和细胞生物学方法研究发病机制。此外，人类原代细胞培养物可作为创建各种转分和转基因培养物的前体6。
然而，与永生化细胞培养物相比，原代细胞的显着缺点是其传代能力有限。因此，我们建议在早期传代阶段（最多15个）使用细胞。较老的文化退化得非常快，失去了它们独特的属性。因此，新获得的原代细胞应保持冷冻以长期储存。
原代细胞培养物易受培养条件的影响。因此，它们通常需要独特的方法和生长条件的优化。特别是，我们实验中使用的人体皮肤原发性成纤维细胞对底物的要求很高。因此，我们根据实验类型使用了各种附加涂层（例如明胶或纤连蛋白）。
细胞细胞骨架决定了细胞的形状、迁移率和运动。细胞骨架的动力学对于相间和有丝分裂中的许多细胞内过程至关重要。特别是，由微管蛋白聚合的细胞骨架是高度动态和极性的结构，能够使运动蛋白介导的定向细胞内转运。微管的末端处于不断的重排中，它们的组装阶段与拆卸阶段交替，这种行为被称为"动态不稳定性"7，8，9。各种相关蛋白质改变了聚合反应的平衡，导致聚合物形成或蛋白质单体形成。微管蛋白亚基的添加主要发生在微管10的正端 。终结合（EB）蛋白家族由三个成员组成：EB1，EB2和EB3。它们充当加端跟踪蛋白（+TIPs），并通过结合和稳定其生长的正端来调节微管的动态特性11。
许多研究使用荧光分子标记的微管蛋白显微注射或转染以及延时成像和视频分析，以 在体外可视化微管 。这些方法可能是侵入性的，对细胞有害，尤其是原代人类细胞。最具挑战性的步骤是找到细胞转染的条件。我们试图在不影响活力和天然细胞形态的情况下达到尽可能高的转染水平。本研究应用经典方法研究健康供体和亨廷顿舞蹈症患者皮肤成纤维细胞微管动力学的差异。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Instrumentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera iXon DU897 EMCCD</td>
			<td>Andor Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5804 R</td>
			<td>Eppendorf Corporate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence filter set HYQ FITC</td>
			<td>Nikon</td>
			<td></td>
			<td>Alternative: Leica, Olympus, Zeiss</td>
		</tr>
		<tr>
			<td>LUNA-II Automated Cell Counte</td>
			<td>Logos Biosystems</td>
			<td>L40002</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope incubator for lifetime filming</td>
			<td>Okolab</td>
			<td>Temperature controller H301-T-UNIT-BL-PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Gas controller CO2-O2-UNIT-BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens CFI Plan Apo Lambda 60x Oil 1.4 (WD 0.13)</td>
			<td>Nikon</td>
			<td></td>
			<td>Alternative: Leica, Olympus, Zeiss</td>
		</tr>
		<tr>
			<td>Widefield fluorescence light microscope Eclipse Ti-E</td>
			<td>Nikon</td>
			<td></td>
			<td>Alternative: Leica, Olympus, Zeiss</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji (Image J version 2.1.0/1.53c)</td>
			<td>Open source image processing software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Alternative: Leica, Olympus, Zeiss</td>
		</tr>
		<tr>
			<td>Additional reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil (Light white oil)</td>
			<td>MP</td>
			<td>151694</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>SPL Lifesciences</td>
			<td>20035</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Dish (glass thickness 170 &#181;m)</td>
			<td>SPL Lifesciences</td>
			<td>211350</td>
			<td>Alternative: MatTek</td>
		</tr>
		<tr>
			<td>Conical Centrifuge tube</td>
			<td>SPL Lifesciences</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic Vials</td>
			<td>Corning-Costar</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tube</td>
			<td>Nest</td>
			<td>615001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultivation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>PanEko</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/62963/62963eq01.jpg" />135</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Media)</td>
			<td>PanEko</td>
			<td>C420<img alt="Equation 2" src="/files/ftp_upload/62963/62963eq02.jpg" /></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#39;s phosphate-salt solution)</td>
			<td>PanEko</td>
			<td>P060<img alt="Equation 2" src="/files/ftp_upload/62963/62963eq02.jpg" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>K053/SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin (bovine skin)</td>
			<td>PanEko</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/62963/62963eq01.jpg" />070</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (1x) + Glutamax</td>
			<td>Gibco</td>
			<td>519850026</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>PanEko</td>
			<td>A063<img alt="Equation 2" src="/files/ftp_upload/62963/62963eq02.jpg" /></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid DNA with EB3-GFP</td>
			<td>Kind gift of Dr. I. Kaverina [Vanderbilt University, Nashville] with permission from Dr. A. Akhmanova 
			[Erasmus University, Rotterdam]</td>
			<td>Stepanova et al., 2003 DOI: 10.1523/JNEUROSCI.23-07-02655.2003</td>
			<td></td>
		</tr>
	</tbody>
</table>

microtubule plus-end dynamics visualization in huntington's disease model based on human primary skin fibroblasts

用荧光标记的感兴趣标记蛋白转染并结合延时视频显微镜是研究细胞骨架动态特性的经典方法。该协议为人原发性成纤维细胞转染提供了一种技术，由于原代细胞培养条件的特殊性，这可能很困难。此外，细胞骨架动态性能维持需要低水平的转染才能获得良好的信噪比，而不会导致微管稳定。重要的是要采取措施保护细胞免受光诱导的应激和荧光染料褪色的影响。在我们的工作过程中，我们测试了不同的转染方法和方案以及不同的载体，以选择适合人类原发性成纤维细胞研究的最佳条件组合。我们分析了生成的延时视频，并使用ImageJ计算了微管动力学。微管在不同细胞部分的正端动力学并不相似，因此我们将分析分为亚组 - 中心体区域，薄片和成纤维细胞的尾部。值得注意的是，该协议可用于患者样本中细胞骨架动力学的 体外 分析，从而为了解各种疾病发展的动力学迈出了下一步。

使用该协议产生的GFP-EB3电影（图1）说明了微管的动态特性。微管参与不同的细胞过程，它们的动态特性影响来自患者活检材料的原代人细胞培养物的各种生命特征（图2）。
以下参数决定了微管的动态不稳定性：生长速率（聚合）和收缩速率（解聚）;灾难的频率（从聚合到解聚的转变）;救援的频率（从解聚到聚合的过渡）;以及?...

Watch this Scientific Journal Video about Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts at JoVE.com

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

该协议专门用于通过EB3蛋白转染进行微管加端可视化，以研究它们在原代细胞培养物中的动态特性。该协议是在从亨廷顿舞蹈症患者获得的人类原发性皮肤成纤维细胞上实施的。

基于人类原发性皮肤成纤维细胞的亨廷顿舞蹈症模型微管加端动力学可视化

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...

亨廷顿舞蹈症HD是一种无法治愈的神经退行性病理学，由编码亨廷顿蛋白的基因突变引起。亨廷顿蛋白主要与囊泡和微管有关，可能参与微管依赖性运输过程。为了研究突变亨廷顿蛋白对微管活力的影响，我们使用EB3蛋白的体内可视化，通过弯曲和稳定生长的正端来调节微管的动态特性。为了将荧光标记的EB3加载到人皮肤成纤维细胞中，我们应用了质粒转染。我们使用原代成纤维细胞培养物从EB3患者的皮肤活检中获得本研究。在几小时内将活检用DMEM培养基送至实验室，补充每毫升青霉素15微克，每毫升链霉素50单位。将活检组织与少量培养基一起放入六厘米培养皿中。使用无菌手术刀，将活检样本切成约0.5至1毫米大小的碎片。将一个，两个获得的碎片放入3.5厘米的培养皿中，并在活检片上放置无菌盖玻片。慢慢加入1.5毫升生长培养基。在CO2培养箱的生长培养基中培养纤维塑料，在5%CO 2中，即7摄氏度，80%湿度。四，七天后，首先是角质形成细胞，然后成纤维细胞开始从组织迁移到培养皿的底部。细胞培养。对于转染可视化，应使用玻璃共聚焦板培养皿，共聚焦培养皿。用蒸馏水制备的高压灭菌的0.1%明胶溶液盖住盘子底部。孵育 15 分钟。准备培养基。充分混合，储存在4摄氏度以上。将培养基加热至37摄氏度，然后加入细胞。在显微镜下评估培养物。取出培养基，并用无菌DPBS洗涤细胞。向细胞中加入一毫升预热的0.25%胰蛋白酶溶液。在显微镜下检查细胞是否完全脱离底物。用一毫升培养基使胰蛋白酶失活。将细胞悬浮液转移到15毫升的锥形管中。将管以200 G离心五分钟。除去上清液，将细胞调色板重悬于一毫升培养基中。计算单元格编号。计算所需的单元格数。用8到15的密度将它们去种子，乘以10到3的幂，经过一厘米。将其平移到培养基的毫升中。从准备接种的培养皿中取出明胶溶液，并立即加入两毫升细胞悬浮液。在CO2培养箱中以37摄氏度培养成纤维细胞。每两、四天刷新一次培养基。对于实验，使用4个，11个传代的细胞。到转染时，汇合处应不超过70，80%在转染前24小时用新鲜的培养基替换培养基。根据细胞接种的面积和密度制备DNA-脂质复合物。脂质体转染剂，并将细胞接种的密度乘以10到4个细胞的幂，用一厘米递过。将三微升商用试剂加入125微升Opti-MEM中，不含抗生素。不要接触管壁，轻轻重悬。稀释质粒DNA，GFP-EB3，将一微克质粒DNA加入125微升OptiMEM中。轻轻重悬。将稀释的质粒DNA加入到稀释的转染试剂的每管中，比例为一比一。孵育 30 分钟。将DNA脂质复合物加入带有细胞的六厘米培养皿中。与十字形摆动混合30秒。用转染剂孵育细胞24小时，然后将培养基换成新鲜培养基。在24和48小时内分析检查后的效率。准备成像。在细胞活体成像之前，将培养基更换为没有PH指示剂染料的培养基，以减少自发荧光。小心地将矿物油涂抹在介质的细胞相上，以完全覆盖介质，将其与外部环境隔离，以减少O2渗透和介质蒸发。使用微距灯和所有图像在60倍，100倍物镜中拍摄，具有高数字光圈拍摄图像。对于在viva观测中，显微镜必须配备培养箱以保持细胞的必要条件，包括击中光学台，并且透镜通过37摄氏度，封闭室供应CO2，以及湿度水平支持。在成像之前，将共聚焦培养皿与细胞一起置于显微镜的支架中。确保碟形天线和相机牢固地固定在支架上，以避免在拍照时漂移。设置成像参数。选择低暴露值，因为载玻片诱导细胞损伤，与氧气空间反应。为了研究人类皮肤成纤维细胞中微管的动力学，我们选择了300毫秒的暴露。专注于感兴趣的对象。对于长时间的延时成像，自动对焦稳定系统，完美的对焦系统是必要的，因为沿设定轴可能会有偏移，因此感兴趣的物体将失焦。根据电池的光敏性和耀斑铬褪色的速率，选择最佳成像条件。由于微管是高度动态的结构，因此可以选择相当短的时间，并且帧速率必须足够高。为了研究皮肤成纤维细胞中的微管动力学，我们使用每秒一帧的频率，持续三到五分钟。当选择下一个物体获取图像时，请远离已经成像的区域，因为在光线的影响下，存在可忽略的光出血。由于我们使用的成像频率相对较高，因此快门在图像之间没有关闭，并且灯在整个成像期间都迟到了，并且褪色增加。选择最佳视频，以直观地研究微管动力学，同时考虑转染质量，微管图像的质量，最佳信噪比以及分析细胞的无漂移。使用选定的视频来研究微管加端动力学，方法是在斐济计划中桁架它们。方案中最关键的步骤是转染，确保足够的蛋白表达。良好的信噪比、无微管光漂白以及无细胞漂移对于成像至关重要。微管动力学的体内可视化提供了关于突变亨廷顿蛋白特性的重要信息。我们的方案可以应用于其他疾病的研究，其病理学涉及细胞骨架的动态特性。

Research

JoVE Journal

Bioengineering

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

三维人体皮肤重建模型：一种工具来研究正常皮肤恶性黑色素瘤进展

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

科研

生物工程

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...