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Recherche en cancérologie

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Published: January 07, 2019
doi:
10.3791/58825
, , , ,
1Genetics and Bioengineering,Yeditepe University, 2National Heart, Lung, and Blood Institute (NHLBI), Sickle Cell Branch,National Institutes of Health (NIH)

Summary

我们提供了基于直接和间接共培养方法评估从牙髓和前列腺癌细胞相互作用中分离出的间充质干细胞的方案。条件介质和反井膜适用于分析间接副胺活性。一起播种不同染色细胞是直接细胞相互作用的合适模型。

Abstract

癌症作为一个多步骤的过程和复杂的疾病, 不仅受单个细胞增殖和生长的调节, 还受肿瘤环境和细胞相互作用的控制。癌症和干细胞相互作用的识别, 包括细胞外环境的变化、身体的相互作用和分泌的因素, 可能有助于发现新的治疗方案。我们结合已知的共培养技术, 建立了间充质干细胞 (mscs) 和癌细胞相互作用的模型系统。在目前的研究中, 牙髓干细胞 (dpscs) 和 pc-3 前列腺癌细胞相互作用的直接和间接共培养技术进行了研究。利用从 dpscc 和0.4μm 孔径反井膜中获得的条件介质 (cm) 研究了副井活性。对不同细胞类型的共培养进行了研究, 以研究细胞直接相互作用。结果表明, cm 能增加前列腺癌细胞培养中细胞增殖, 减少细胞凋亡。cm 和经井系统都提高了 pc-3 细胞的细胞迁移能力。用不同的膜染料染色的细胞被播种到同一培养容器中, dpscs 在这种直接共培养条件下参与了与 pc-3 细胞的自组织结构。总体而言, 结果表明, 共培养技术可用于癌症和 msc 相互作用作为一个模型系统。

Introduction

间充质干细胞 (mscs) 具有分化能力, 有助于骨髓、软骨、肌肉、韧带、肌腱和脂肪等间充质组织的再生, 几乎从成人体内的所有组织中分离出 1,2. 除了在慢性炎症或损伤的情况下通过产生常驻细胞来提供组织稳态外, 它们还产生重要的细胞因子和生长因子, 以协调血管生成、免疫系统和组织重塑3。骨髓间充质干细胞与肿瘤组织的相互作用尚不清楚, 但积累的证据表明, 骨髓间充质干细胞可能促进肿瘤的发生、进展和转移4。

骨髓间充质干细胞对受伤或长期发炎区域的归巢能力使其成为干细胞治疗的宝贵候选。然而, 癌症组织, “永远不会愈合伤口”, 也释放炎性细胞因子, 亲血管生成分子, 和重要的生长因子, 吸引骨髓间充质干细胞到癌变区 5.虽然有有限的报告显示骨髓间充质干细胞对癌症生长的抑制作用 6,7, 他们的癌症进展和转移促进的影响已被广泛报道8。骨髓间充质干细胞以不同的方式直接或间接地影响致癌, 包括抑制免疫细胞、分泌支持癌细胞增殖和迁移的生长因子细胞因子、增强血管生成活性和调节上皮间充质转变 (emt)9,10。肿瘤环境由多种细胞组成, 包括与癌症相关的成纤维细胞 (caf) 和肌成纤维细胞、内皮细胞、脂肪细胞和免疫细胞11。其中, caft 是肿瘤区最丰富的细胞类型, 分泌各种促进癌症生长和转移的趋化因子8。研究表明, 骨髓源性骨髓间充质干细胞可在肿瘤基质12中分化为 caf。

牙髓干细胞 (dpscs), 被描述为第一个牙科组织衍生的骨髓间充质干细胞由 gronthos等人.13在 2000年, 然后被广泛调查的其他 14,15, 表达多能标记, 如oct4, sox2, 和nanog16 , 并可以分化为各种细胞链17。基因和蛋白质表达分析证明, dpsc 产生的生长因子/细胞因子水平与其他骨髓间充质干细胞相当, 如血管内皮生长因子 (vegf)、血管生成素、成纤维细胞生长因子 2 (fgf2)、白细胞介素-4 (il-4)、il-6、il-10、和干细胞因子 (scf), 以及 fms 样酪氨酸激酶-3 配体 (flt-3l), 可能促进血管生成, 调节免疫细胞, 并支持癌细胞增殖和迁移18,19,20.虽然骨髓间充质干细胞与癌症环境的相互作用已在文献中得到了充分的记录, 但 dpscs 与癌细胞之间的关系尚未得到评估。在本研究中, 我们建立了高度转移性前列腺癌细胞系 pc-3 和 dpscs 的共培养和条件培养基治疗策略, 提出了牙科骨髓间充质干细胞机制在癌症进展和转移中的潜在作用。

Protocol

经机构道德委员会批准后, 获得了患者的书面知情同意。 1. dpsc 隔离和培养 从17至20至20至15毫升的年轻成年人身上获得的智齿含有完整的 dulbecco 的修饰鹰培养基 (dmem) [低葡萄糖 dmem 培养基, 辅以10% 的胎牛血清 (fbs) 和1% 青霉素/两性霉素 (psa) 溶液], 切除后8小时内。在转移过程中保持组织材料的低温 (4°c), 以避免潜在的细胞死亡。 通过无菌拔模从牙齿中心小心地?…

Representative Results

图 1描述了在培养条件下 dpscs 的一般 msc 特征。dpsc 在电镀后发挥成纤维细胞样细胞形态 (图 1b)。msc 表面抗原 (cd29、cd73、cd90、cd105 和 cd166) 表达强烈, 而造血标志物 (cd34、cd45 和 cd14) 为阴性 (图 1c)。在 dpsc 培养中观察到与骨、软骨和脂肪基因分化有关的形态和分子水平的变化, 然后是分化鸡尾酒的应用<stron…

Discussion

骨髓间充质干细胞对肿瘤环境的贡献受到多种相互作用的调节, 包括干细胞与癌细胞之间的混合细胞融合、昆虫病或细胞因子和趋化因子活性.结构组织、细胞相互作用和分泌因素决定了癌细胞在肿瘤促进、进展和转移到周围组织方面的行为。需要适当的体外模型系统来研究住院细胞群体相互作用背后的机制, 以了解细胞通信促进癌症进展和转移。

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Divulgations

The authors have nothing to disclose.

Acknowledgements

这项研究得到了叶迪特佩大学的支持。本文中使用的所有数据和数字都是以前公布的。

Materials

DMEM Invitrogen 11885084 For cell culture
FBS Invitrogen 16000044 For cell culture
PSA Lonza 17-745E For cell culture
Trypsin Invitrogen 25200056 For cell dissociation
PBS Invitrogen 10010023 For washes
Dexamethasone Sigma D4902 Component of differentiation media
β-Glycerophosphate Sigma G9422 Component of osteogenic differentiation medium
Ascorbic acid Sigma A4544 Component of osteo- and chondro-genic differentiation medium
Insulin-Transferrin-Selenium (ITS −G) Invitrogen 41400045 Component of chondrogenic differentiation medium
TGF-β Sigma SRP3171 Component of chondrogenic differentiation medium
Insulin Sigma I6634 Component of adipogenic differentiation medium
Isobutyl-1-methylxanthine (IBMX) Sigma I7018 Component of adipogenic differentiation medium
Indomethacin Sigma I7378 Component of adipogenic differentiation medium
MTS Reagent Promega G3582 Cell viability analyses
TUNEL Assay Sigma 11684795910 Apoptotic analyses
24-well plate inserts Corning 3396 For trans-well migration assay
PKH67 Sigma PKH67GL For co-culture cell staining
PKH26 Sigma PKH26GL For co-culture cell staining
Paraformaldehyde Sigma P6148 For cell fixation
von Kossa Kit BioOptica 04-170801.A For cell staining (differentiation)
Alcian blue Sigma A2899 For cell staining (differentiation)
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Tags

Keywords: Mesenchymal Stem CellsCancer CellsCo-cultureCell InteractionsStem Cell IsolationCell CultureCell MorphologySurface MarkersCell FixationCell Staining
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Doğan, A., Demirci, S., Apdik, H., Apdik, E. A., Şahin, F. Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions. J. Vis. Exp. (143), e58825, doi:10.3791/58825 (2019).
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