这项工作得到了国家卫生和医学研究理事会 (澳洲) 给 H-爵士的赠款的支持。TMBW 是澳大利亚研究生奖的接受者澳大利亚政府和 Ann 亨德森的顶部奖学金从澳大利亚扶轮健康与扶轮 Templestowe 和用餐的治疗。

所有动物实验都是由墨尔本大学动物伦理委员会批准的。
注: 腺病毒载体的序列, 构造和生成协议 pad-巨细胞病毒-Td-汤姆, pad cmv-GFP和 pad-CAGA12-卢克/Td-汤姆以前被描述了11,12, 13。所有的向量都是商用的。
1. 使用50% 组织培养感染剂量的病毒效价测定 (TCID50</s...

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我们开发了一种技术, 允许实时成像的 TGF-β/Smad3 信号在单个活细胞。利用这一新方法, 我们以前已经确定了一个子群体的细胞与动态 TGF-β/Smad3 转录活动, 与增强入侵和迁移8。该方法改进了传统的 TGF β信号检测, 如 Smad3 磷酸化和 TGF β靶向基因表达的西方印迹, 通过捕获肿瘤人群中 TGF-β/Smad3 信号的异质性, 突出单细胞生物学的重要性。此外, 单细胞成像的替代方法, 如核易位可以?...

转化生长因子β (TGF β) 是人类发展中的一项重要的细胞因子, 它通过由 II 型和 I 型受体1组成的 heterodimeric 复合物发出信号。对 II. 型受体的结合导致 I 型受体的招募和磷酸化, 进而 phosphorylates 下游 Smad2/3 蛋白2,3。这些活化 Smad2/3 蛋白绑定到 Smad4, 形成一个复杂的 translocates 进入细胞核和调控基因转录4。在恒定的情况下 TGF β/Smad 信号被严密地调控;然而, 在许多疾病中, 信号通路被解除管制, 并且经常抗原导致疾病的进展5,6,7。最近的研究表明, 细胞对 TGF β的反应是异质的, TGF β/Smad 活性细胞的亚群是以时间依赖性的方式8,9负责生物功能。TGF β/Smad 信号的常见细胞分析涉及使用固定端点化验, 只提供细胞活动的快照, 而且通常定量平均 TGF β/Smad 效应10。然而, 这些方法可能不能准确地反映 TGF-β/Smad 信号在疾病进展过程中生理状态下的分子行为。基于图像的活细胞分析捕捉细胞和生物过程的动态, 既具有空间又有时间的理解。
我们的目标是开发一种灵敏的高通量方法用于活细胞成像的 TGF β/Smad 信号使用腺病毒试剂。在这里, 我们感染了人类乳腺癌细胞系 MDA-MB-231 与腺病毒表达 Smad3 CAGA 的主题结合序列和荧光素酶 (卢克) 或 td-番茄 (td-汤姆) 报告基因。腺病毒报告系统为质粒的引入提供了一种快速、廉价的方法, 可导致癌细胞株感染率100%。腺病毒报告系统也已成功地应用于细胞系难以染与常规质粒11。在本协议中, 我们将描述一个可重现和无创的过程, 以实现 TGFβ/Smad 信号通路的活体细胞成像在体内和体外。

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>1881024</td>
			<td>Warm in 37 &#176;C waterbath before use</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Scientifix Life</td>
			<td>FBS500-S</td>
			<td>Heat inactivated before use</td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-&#946;1</td>
			<td>PEPROTECH</td>
			<td>100-21</td>
			<td>Aliquot in DDW to make final concentration at 10 mg/mL</td>
		</tr>
		<tr>
			<td>Hoechst-33258</td>
			<td>Tocris Bioscience</td>
			<td>5117</td>
			<td>Dilute in to PBS to make final concentration at 1 &#956;L/mL</td>
		</tr>
		<tr>
			<td>Luciferase Reporter Assay Kit</td>
			<td>Promega</td>
			<td>197897</td>
			<td>Dilute 5x in PBS before use</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>9100-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast fluorescence microscopy</td>
			<td>OLYMPUS</td>
			<td>IX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoGl Luciferin</td>
			<td>Promega</td>
			<td>P1041</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina III In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>CLS136334</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA (10x)</td>
			<td>ThermoFisher</td>
			<td>15400-054</td>
			<td>Diltue to 0.05% (1x) in PBS</td>
		</tr>
		<tr>
			<td>Cell Culture Lysis 5x Reagent</td>
			<td>Promega</td>
			<td>E153A</td>
			<td>Dilute to 1x in DDW</td>
		</tr>
		<tr>
			<td>10% Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>F5554-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293A</td>
			<td>ThermoFisher</td>
			<td>R70507</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>CRM-HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>PRKDC-SCID</td>
			<td>Animal Resources Centre</td>
			<td>SCIDF6</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Chem-supply</td>
			<td>EA043-10L-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Refresh Night Time</td>
			<td>Allergan</td>
			<td>1750D</td>
			<td>Lubricating Eye Ointment</td>
		</tr>
		<tr>
			<td>Solution</td>
			<td></td>
			<td></td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td>NaH2PO4.2H2O (4 mM); NaHPO4 (16 mM); NaCl (0.12M)</td>
		</tr>
		<tr>
			<td>FBS-DMEM&#160;</td>
			<td></td>
			<td></td>
			<td>5% heat inactivated FBS; 10 &#956;g/mL penicillin; 100 &#956;g/mL streptomycin</td>
		</tr>
	</tbody>
</table>

live cell imaging of the tgf-	&beta;/smad3 signaling pathway in vitro and in vivo using an adenovirus reporter system

转化生长因子β (TGF) 信号调节细胞稳态所需的许多重要功能, 在许多疾病, 包括癌症中普遍发现抗原。TGF β在晚期癌症进展中与转移有强烈牵连, 激活了迁移和侵袭性肿瘤细胞的一个子集。目前的信号通路分析方法侧重于端点模型, 通常试图测量生物事件后的信号, 不反映疾病的渐进性质。在这里, 我们展示了一个新的腺病毒记者系统特定的 TGF-β/Smad3 信号通路, 可以检测转录激活的活细胞。利用Ad CAGA12-Td-汤姆记者, 我们可以在24 小时内达到 100% MDA-MB-231 细胞感染率。使用荧光记者可以实时地对活单细胞进行成像, 直接识别转录活性细胞。用 TGF β刺激感染细胞, 只显示转录活性和参与特定生物学功能的细胞子集。这种方法允许在单个细胞水平高特异性和敏感性, 以增强对与 TGF β信号的生物学功能的认识。Smad3 转录活动也可以在体内实时报告通过应用一个Ad CAGA12-卢克记者。CAGA12-卢克可以用与传统稳定转染的荧光素酶细胞系相同的方式来测量。Smad3在体内植入细胞的转录活性可以通过常规的 IVIS 影像分析, 并在肿瘤进展过程中进行监测, 为 TGF β信号通路的动力学提供独特的洞察力。我们的协议描述了一个有利的报告传递系统, 允许快速高通量成像的活细胞信号通路的体外和体内。这种方法可以扩展到一系列基于图像的检测, 并呈现为基础生物学和治疗发展的敏感和可重复的方法。

活体单细胞成像的体外研究
为了准确评估 TGF-β/Smad 信号在单细胞中的活化作用, 使用腺病毒试剂, 首先确定每一个细胞系的最佳感染多样性 (语言) 是很重要的。当100% 的细胞对腺病毒感染有阳性反应时, 不存在病变或细胞毒性作用, 就确定了最佳的教学语言。为了确定这一点, 我们使用了一个组成性活跃的Ad-巨细胞病毒-T...

Watch this Scientific Journal Video about Live Cell Imaging of the TGF-	ÃŽÂ²/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System at JoVE.com

Live Cell Imaging of the TGF-	ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

腺病毒报告系统 TGF β/Smad3 信号通路的活体细胞成像

在这里, 我们提出了一个使用腺病毒记者系统的 TGF-β/Smad3 信号活动的活细胞成像协议。该系统实时跟踪转录活性, 可用于体外和活体动物模型中的单细胞.

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Research

JoVE Journal

Cancer Research

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

科研

癌症研究

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

In Vitro Assay to Study Tumor-macrophage Interaction

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...