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Pesquisa do Câncer

固体肿瘤细胞系侧面人口分析

Published: February 23, 2021
doi:
10.3791/60658
, , , ,
1School of Medicine,Nankai University, 2Tianjin Key Laboratory of Tumour Microenvironment and Neurovascular Regulation, 3Collaborative Innovation Center for Biotherapy,Nankai University

Summary

提出了一种方便、快速、经济的方法，测量固体肿瘤细胞系中侧群细胞的比例。

Abstract

癌症干细胞（CSC）是肿瘤生长、转移和复发的重要原因。CSC的分离和鉴定对肿瘤研究具有重要意义。目前，几种技术用于从肿瘤组织和肿瘤细胞系中识别和净化CSC。侧群 （SP） 细胞的分离和分析是两种常用的方法。这些方法依赖于 CSC 快速排出荧光染料（如 Hoechst 33342）的能力。染料的流出与 ATP 绑定盒式 （ABC） 运输机相关联，可由 ABC 运输器抑制剂抑制。介绍了用Hchst 33342染色培养肿瘤细胞的方法，并通过流动细胞测量分析其SP细胞的比例。此检测方便、快速且经济高效。此测定中生成的数据有助于更好地了解基因或其他细胞外和细胞内信号对肿瘤细胞干细胞特性的影响。

Introduction

癌症干细胞（CSC）是具有自我更新能力和多分化潜力的细胞子集，在肿瘤生长、转移和复发1、2中起着至关重要的作用。目前，CSC已确定存在于各种恶性肿瘤中，包括肺癌、脑癌、胰腺癌、前列腺癌、乳腺癌和肝癌3、4、5、6、7、8、9。这些肿瘤中CSC的鉴定主要基于表面标记蛋白的存在，如CD44、CD24、CD133和Sca-19、10的高和/或低表达，但迄今尚未报告能够区分CSC与非CSC的独特标记。目前，用于识别和净化肿瘤组织或肿瘤细胞系中的 CSC 的几种技术。这些技术是根据 CSC 的特定属性设计的。其中，侧群（SP）细胞的测定和排序是两种常用的方法。

SP细胞最初是由古德尔等人11日发现的，当时它们特征是小鼠骨髓细胞中的造血干细胞。当小鼠骨髓细胞被贴上荧光染料Hhchst 33342的标签时，一小群Hchst 33342模糊染色细胞出现在流动细胞测定测量的二维点图中。Hoechst 33342 是一种 DNA 结合染料，至少有两种绑定模式，导致不同的光谱特征。同时以两个波长查看荧光发射时，可以显示多个种群12。在他们的测定中，Hoechst 33342 在 350 nm 时兴奋不已，荧光是使用 450/20 nm 带通 （BP） 滤光片和 675 nm 边缘滤光长通 （EFLP）11来测量的。与整个骨髓细胞群相比，这组细胞富含称为SP细胞11的造血干细胞。SP细胞能够快速驱逐霍赫斯特33342。这种染料的流向与ATP绑定盒式磁带（ABC）运输机13有关，可以抑制一些代理，如富米特雷莫金C14，维拉帕米尔和雷塞平15，16。之后，在各种组织、器官、肿瘤组织和肿瘤细胞系17、18、19中检测出不同比例的SP细胞。这些SP细胞具有干细胞的许多特征17，19。

本手稿描述了霍奇斯特33342对培养肿瘤细胞的标记和染色，以及通过流动细胞学对SP细胞的分析。此外，还显示了使用此方法优化的 Hoechst 33342 浓度和特定肿瘤细胞系的适当阻滞剂选择。最后，证明了干细胞促进或抑制信号对肿瘤细胞SP比例的影响。实验实例表明，对SP的分析可用于探索各种信号的影响，如基因表达、小抑制剂、活化剂、细胞因子和化疗因子，对肿瘤茎的作用。与其他分离和纯化 CSC 的方法（如 CD44+/CD24） 种群排序、醛脱氢酶 （ALDH） 分析和肿瘤球体形成检测）相比，该方法更容易操作，且具有成本效益。

Protocol

1. 细胞准备 细胞消化和中和 种子肿瘤细胞（如MDA-MB-231细胞）在6井板，并在37°C孵化器中培养他们提供5%的二氧化碳。 当细胞密度达到90%左右时，收获细胞。彻底吸气培养介质，用3mL磷酸盐缓冲盐水（PBS）清洗细胞2倍。注意：为了检查信号通路抑制剂（例如FRA1抑制剂）或活化剂（例如STAT3活化剂）对肿瘤细胞干细胞特性的影响，肿瘤细胞在6个井板中播种，并在收…

Representative Results

根据这种方法进行了四次实验性SP分析。在第一个，我们检测到SP细胞在MDA-MB-231中的比例，这是人类乳腺癌细胞系的三重阴性，在正常条件下。在细胞计数后，Hoechst 33342 被添加到一个包含 1 x 106细胞的管中，最终浓度为 3 μg/mL。Reserpine 和 Hoechst 33342 分别添加到另一根管中，最终浓度分别为 40μM 和 3μg/mL。PI 被添加到两个管中。FSC-A（X轴）与PI-A（Y轴）的点图显示了三个种群：1） 代表死细…

Discussion

对于 SP 分析，需要牢记几个要点。首先是为每个细胞系选择适当的阻滞剂，如维拉帕米尔或雷塞平，因为 SP 细胞的”门”位置根据添加阻止器后大量 SP 细胞消失的位置确定。对于MDA-MB-231细胞系，雷瑟平工作得很好。然而，对于其他细胞系，不同的阻滞剂可能效果更好。

第二个是霍赫斯特33342的浓度。有代表性的数据显示，随着霍赫斯特33342的染色浓度降低，SP细胞的百分比增?…

Declarações

The authors have nothing to disclose.

Acknowledgements

本工作由中国自然科学基金委员会81572599、81773124、81972787资助：天津市自然科学基金（中国） 19JCYBJC27300：天津市人民医院 – 南开大学合作研究资助2016rmnk005;中央大学基础研究基金、南开大学63191153。

Materials

6 well cell culture plate CORNING 3516 9.5 cm2 (approx.)
Colivelin MCE HY-P1061A Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-Pro-Ala-Gly-Ala-Ser-Arg-Leu-Leu-Leu-Leu-Thr-Gly-Glu-Ile-Asp-Leu-Pro
Fetal bovine serum (FBS) Biological Industries (BIOIND) 04-001-1ACS
Flow cytometer BD Biosciences BD LSRFortessa
Flow cytometer software BD Biosciences FACSDiva
Flow cytometry analysis software BD Biosciences FlowJo
Hoechst33342 Sigma-Aldrich B2261 bisBenzimide H 33342 trihydrochloride
Polystyrene round bottom test tube CORNING 352054 12 x 75 mm, 5mL
Propidium iodide (PI) Sigma-Aldrich P4170 3,8-Diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide
Reserpine Sigma-Aldrich 83580 (3β, 16β, 17α, 18β, 20α)-11,17-Dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester
SKLB816 Provided by Dr. Shengyong Yang, Sichuan University
Trypsin-EDTA (0.25%), phenol red Gibco 25200072
Verapamil hydrochloride Sigma-Aldrich V4629 5-[N-(3,4-Dimethoxyphenylethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2-isopropylvaleronitrile hydrochloride
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Tags

Side PopulationCancer Stem CellsTumor Cell LinesHoechst 33342Flow CytometrySP AnalysisStemness PropertiesCell CultureCell Preparation
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Dong, X., Wei, Y., Xu, T., Tan, X., Li, N. Analysis of Side Population in Solid Tumor Cell Lines. J. Vis. Exp. (168), e60658, doi:10.3791/60658 (2021).
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