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腺病毒报告系统 TGF β/Smad3 信号通路的活体细胞成像

Published: July 30, 2018
doi:
10.3791/57926
, , ,
1Department of Surgery (RMH),University of Melbourne

Summary

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.

Abstract

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。

Introduction

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。

Protocol

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…

Representative Results

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…

Discussion

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?…

Declarações

The authors have nothing to disclose.

Acknowledgements

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。

Materials

DMEM ThermoFisher 1881024 Warm in 37 °C waterbath before use
Foetal Bovine Serum Scientifix Life FBS500-S Heat inactivated before use
Recombinant Human TGF-β1 PEPROTECH 100-21 Aliquot in DDW to make final concentration at 10 mg/mL
Hoechst-33258 Tocris Bioscience 5117 Dilute in to PBS to make final concentration at 1 μL/mL
Luciferase Reporter Assay Kit Promega 197897 Dilute 5x in PBS before use
Luminometer Promega 9100-002
Phase contrast fluorescence microscopy OLYMPUS IX50
Centrifuge eppendorf 5810 R
VivoGl Luciferin Promega P1041
IVIS Lumina III In Vivo Imaging System PerkinElmer CLS136334
0.5% Trypsin-EDTA (10x) ThermoFisher 15400-054 Diltue to 0.05% (1x) in PBS
Cell Culture Lysis 5x Reagent Promega E153A Dilute to 1x in DDW
10% Formalin Sigma-Aldrich F5554-4L
HEK 293A ThermoFisher R70507
MDA-MB-231 ATCC CRM-HTB-26
PRKDC-SCID Animal Resources Centre SCIDF6
Matrigel Corning 354234
Isoflurane Zoetis 26675-46-7
Ethanol Chem-supply EA043-10L-P
Refresh Night Time Allergan 1750D Lubricating Eye Ointment
Solution Composition
Phosphate-Buffered Saline (PBS) NaH2PO4.2H2O (4 mM); NaHPO4 (16 mM); NaCl (0.12M)
FBS-DMEM  5% heat inactivated FBS; 10 μg/mL penicillin; 100 μg/mL streptomycin
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Tags

Keywords: Live Cell ImagingTGF-β/Smad3 Signaling PathwayAdenovirus Reporter SystemCancer Cell LinesBreast CancerWound Healing AssayMDA-MB-231 CellsAdenovirus InfectionFluorescence MicroscopyGFPTd-TomatoTGF-betaImageJ
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Chen, H., Ware, T. M., Iaria, J., Zhu, H. Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System. J. Vis. Exp. (137), e57926, doi:10.3791/57926 (2018).
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