转化生长因子β信号诱导内皮-间质转移的分子分析
Summary
本文介绍了一种体外诱导内皮-间质转移 (EndMT) 的方法, 该协议对于研究 EndMT 中涉及的细胞信号通路是有用的。在该实验模型中, EndMT 是由 TGF β在 MS-1 内皮细胞中的治疗引起的。
Abstract
内皮细胞表型可塑性是心血管系统发展、心血管疾病以及与器官纤维化相关的各种条件的基础。在这些条件下, 分化的内皮细胞获得间充质样表型。这一过程称为内皮-间质转移 (EndMT), 其特点是下调的内皮标记, 上调的间充质标记, 和形态学的变化。EndMT 是由几种信号通路引起的, 包括转化生长因子 (TGF) β、Wnt 和凹槽, 并由类似于上皮-间质转移 (急诊) 的分子机制所调控, 对肠、组织纤维化和肿瘤转移。了解 EndMT 的机制对于发展针对 EndMT 的诊断和治疗方法非常重要。EndMT在体外的鲁棒诱导是一种常见的基因表达特征, 识别 druggable 分子机制和 EndMT 调节剂的筛查。在这里, 我们描述了一种诱导 EndMT 的体外方法。MS-1 小鼠胰腺微血管内皮细胞在长时间暴露于 TGF β后 EndMT, 显示上调的间充质标志物和形态学变化以及诱导多种炎症趋化因子和细胞因子。microRNA (miRNA) 调制的分析方法也包括在内。这些方法为调查 EndMT 的机制和 miRNAs 对 EndMT 的贡献提供了一个平台。
Introduction
内皮-间质转移 (EndMT) 是一种分化的内皮细胞经历多种分子变化的过程, 导致成纤维细胞样间质细胞1。EndMT 最初被描述作为内皮细胞变革在心脏的发展期间2,3。在早期心脏发育中, 心脏管由内心内膜和外心肌组成。这两层被一层细胞外基质所分离, 称为心脏果冻。胚胎心内膜细胞, 获得内皮细胞标志物, 转运到间充质细胞, 侵入底层的心脏果冻, 促进心脏垫的形成, 为房室瓣膜和隔膜提供基础和半月形阀门。此外, EndMT 已被认为是毛细血管和血管平滑肌细胞在其他胚胎血管系统的来源, 包括冠状动脉血管, 腹主动脉, 肺动脉4,5,6。此外, EndMT 与生理血管生成7有牵连。
积累的证据表明, EndMT 也参与多种心血管疾病和其他疾病1,8。EndMT 相关的条件包括血管钙化, 动脉粥样硬化, 肺动脉高压, 海绵状畸形, 器官纤维化, 静脉移植重塑, 移植肾功能障碍和癌症8, 9,10,11,12,13,14,15,16,17, 18. 最近的一份报告指出, 几种分子 EndMT 标记物可以作为诊断和预测肾脏移植肾功能障碍的一种工具,17。EndMT 相关的细胞信号通路的调制已被证明可以改善多种疾病的条件, 包括心脏纤维化和静脉移植重塑的动物模型8,15。因此, 了解 EndMT 所依据的机制对于制定针对 EndMT 的诊断和治疗策略是很重要的。
EndMT 的特点是细胞连接的丧失, 迁徙潜能的增加, 内皮特异基因的下调, 如 VE 钙粘蛋白, 上调间充质基因, 包括α平滑肌肌动蛋白 (α SMA)。此外, EndMT 和上皮间质转移 (急诊室), 一个类似的过程, 将上皮细胞转化为间充质细胞, 与各种细胞外基质成分的改变产生相关, 这可能有助于发展组织纤维化8,19。
最近, EndMT 的几项体外研究已经阐明了 EndMT15、20的分子机制的细节。EndMT 是由各种信号通路, 包括转化生长因子 (TGF) β, Wnt, 和缺口1诱导。其中, TGF β在急救和 EndMT 诱导中起着举足轻重的作用。在 EndMT, 长期暴露于 TGF β的结果 EndMT 在各种内皮细胞, 而短期暴露似乎不足21。我们这里描述了一个简单的 EndMT 诱导协议, 其中英里斯文 1 (MS-1) 小鼠胰腺微血管内皮细胞在长时间接触 TGF β20后,体外EndMT。在该模型中, 可以进行多种下游分析, 以研究 EndMT 的标志性特征, 包括形态学变化、内皮标记下调、间充质标志物上调和炎症基因骨架重排, 胶原凝胶收缩。
MicroRNAs (miRNAs) 是 22 nt 小规 rna, 直接转录后水平压制各种 mRNA 目标22,23。通过种子序列介导的目标识别, miRNAs 抑制数以百计的靶基因, 调节细胞分化、增殖和运动等多种细胞功能。这也是对急诊医师和 EndMT 的监管, 并且有几个 miRNAs 被报告为急诊医师和 EndMT24,25的监管者。本文提出的 EndMT 模型可以很容易地结合 miRNA 调制程序来测试 miRNAs 在 EndMT 中的作用。本文综述了 TGF β诱导 EndMT 在 MS-1 细胞中的实验方法, 并对其他内皮细胞中 TGF β的 EndMT 诱导条件进行了比较。
Protocol
Representative Results
Discussion
据报道, 活化 Ras 和 TGF β治疗24小时诱导 EndMT 在 MS-1 细胞, 而 TGF β单独未能诱导 EndMT 在这短短的时间21。我们一贯地观察到, TGF β在 MS-1 细胞中的长时间治疗 (48–72 h) 后, 极大地诱发了 EndMT20。EndMT 在 TGF β (2–6天) 在各种内皮细胞如人脐静脉内皮细胞 (HUVEC)、人皮肤微血管内皮细胞 (HCMEC)、人冠状动脉内皮细胞等的长期治疗后反复观察 (HCEC), 小鼠肺内皮细胞 (《电…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢玉米 Borok 和晃平 Miyazono 为编写手稿提出建议。H.I.S. 和 M.H. 得到了上原纪念基金会研究金的支持, H.I.S. 得到了留学 Hayaishi 纪念奖学金的支持。这项工作得到了武田科学基金会 (犍为县) 的资助。
Materials
| MS-1 cells | American Type Culture Collection | CRL-2279 | |
| MEM-alpha | Thermo Fisher Scientific | 32571036 | |
| TGF-beta2 | R&D | 302-B2-002 | |
| 4 well Lab-Tek II Chamber Slide | Thermo Fisher Scientific | 154526 | |
| Y-27632 | Sigma-Aldrich | Y0503 | |
| Blocking One | nacalai tesque | 03953-95 | |
| phalloidin-tetramethylrhodamine B isothiocyanate | Sigma-Aldrich | P1951 | |
| TOTO-3 iodide | Thermo Fisher Scientific | T3604 | |
| VE cadherin monoclonal antibody (BV13) | Thermo Fisher Scientific | 14-1441-82 | |
| alpha-SMA Cy3 monoclonal antibody (1A4) | Sigma-Aldrich | C6198 | |
| Alexa Fluor 488 goat anti-mouse IgG (H+L) | Thermo Fisher Scientific | A-11001 | |
| Cover slip | Thermo Fisher Scientific | 174934 | |
| Collagen solution | Nitta gelatin Inc. | Cellmatrix I-P | |
| Collagen dilution buffer | Nitta gelatin Inc. | Cellmatrix I-P | |
| LNA miRNA inhibitor | EXIQON | miRCURY LNAmicroRNA Power Inhibitor (Negative Control B and target miRNA) | |
| synthetic miRNA duplex | Qiagen | miScript miRNA Mimic | |
| Lipofectamine RNAiMAX | Thermo Fisher Scientific | 13778030 | |
| Lipofectamine 2000 | Thermo Fisher Scientific | 11668027 | |
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