Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

Lukasz Bozycki; Magdalena Komiazyk; Saida Mebarek; Rene Buchet; Slawomir Pikula; Agnieszka Strzelecka-Kiliszek

doi:10.3791/57423

JoVE Journal > Chemistry

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화학

透射电镜 hFOB 1.19 和 Saos-2 细胞产生的能量色散 X 射线显微分析

Published: June 24, 2018

doi:

10.3791/57423

Lukasz Bozycki, Magdalena Komiazyk, Saida Mebarek^3,4,5,6, Rene Buchet^3,4,5,6, Slawomir Pikula, Agnieszka Strzelecka-Kiliszek

¹Laboratory of Lipid Biochemistry, Nencki Institute of Experimental Biology,Polish Academy of Sciences, ²Université de Lyon, ³Université Lyon 1, ⁴L’insitut National des Sciences Appliquées (INSA) de Lyon, ⁵Ecole Supérieure de Chimie Physique Electronique (CPE) Lyon, ⁶Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR),L’institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS)

Summary

我们提出一个协议, 以比较的矿物质在囊泡释放的两个人骨细胞系: hFOB 1.19 和 Saos-2。利用茜素红 s (AR) 染色、紫外 (紫外) 光可视化、透射电镜 (TEM) 成像和能量色散 X 射线显微分析 (EDX) 对其成矿剖面进行了研究。

Abstract

本视频介绍了利用透射电镜与能量色散 X 射线微分析 (TEM-EDX) 比较的矿物质在囊泡释放的两个人骨细胞线: hFOB 1.19 和 Saos-2。这些细胞系, 经抗坏血酸 (AA) 和β甘油 (β GP) 治疗后, 经过完全成骨分化, 从增殖到成矿, 产生基质泡 (mv), 触发磷灰石成核在细胞外基质 (ECM)。

基于茜素红 s (AR) 染色, 利用透射电镜成像和 EDX 定量和离子图谱对细胞裂解物中的矿物质组成进行分析, 并对其进行了研究, 得出了 Saos-2 和成骨肉瘤hFOB 1.19 细胞显示出明显的矿化剖面。Saos-2 细胞比 hFOB 1.19 细胞矿化更有效, 并产生较大的矿床, 在紫外光下不可见, 但与羟基磷灰石 (HA) 相似, 因为它们有更多的钙和 F 替代物。

利用这些技术得到的结果使我们得出结论, 矿化过程的不同取决于细胞类型。我们建议, 在细胞层面上, 囊泡的起源和性质预先确定了矿物质的类型。

Introduction

骨是由两部分组成的结缔组织: 有机 (细胞和胶原纤维) 和矿物质 (钙和磷酸盐化合物)。骨中主要矿物成分为磷灰石¹。不同类型的成矿能力的细胞在骨 (成骨细胞), 在牙齿 (成牙本质细胞) 和软骨 (软骨细胞) 通过产生细胞外基质 (ECM) 和释放基质的蛋白质来调节矿化的初始步骤囊泡 (mv) (图 1)。100-300 毫微米直径囊泡, 积累钙和磷酸促进磷灰石成核, 随后绑定到胶原蛋白²^,³。然后, 磷灰石释放到胞外培养基中。磷灰石继续与胶原纤维接触, 形成骨基质。矿化由 P_i和钙^{2 +}在胞外培养基中的恒定供给维持。最近发布的一些数据支持我们的模型⁴^,⁵。软组织在生理条件下不矿化。然而, 异位钙化可能发生在病理条件下, 如血管钙化³。获得成骨细胞表型的血管细胞可产生诱导磷灰石的核化, 并在血管壁内侧和内膜层启动成矿作用。由于异位钙化类似于正常的软骨矿化³, 了解骨细胞和软骨细胞成矿作用的分子机制, 应为软组织异位钙化提供一些线索。形成。

骨骼组织的发育受各种酶、生长因子、成矿作用的促进剂或抑制剂的调节。组织非特异性碱性磷酸酶 (TNAP) 的拮抗作用 (图 1) 和 ectonucleotide 焦磷酸酶/磷酸二酯化 I (NPP1), 连同锚蛋白 (谢谢), 控制无机焦磷酸盐 (PP_I) 浓度⁶. PP_i, 一种有效的 HA 形成抑制剂, 由 TNAP 水解;NPP1 水解核苷酸 triphosphates 形成 pp_i , 而谢谢出口 pp_i从细胞到 ECM。Pi/PPi 比率可能调控磷灰石形成⁷^,⁸以可能的病理后果⁹。

MV 膜富含离子传输蛋白, 在核过程中促进了钙和磷酸盐的初始沉淀 (图 1)。磷酸盐传送器 1 (坑) 帮助合并 P_i生成在 perivesicular 空间入¹⁰^,¹¹。Annexins 可能涉及 Ca^{2 +}的结合和运输, 以及在 MV 腔¹²^,¹³中启动矿化的生物物理过程中。我们赞成早些时候提出的假说, 即在其在 ECM¹⁴^、¹⁵中传播之前, 在 MV 内的磷灰石内部成核的胞质囊泡中的矿化。体外建模证实了从 PS 和 AnxA5¹⁶的 proteoliposomes 中诱导的钙^{2 +}/P_i复合物的形成。这可能表明, 钙^{2 +}, P_i, AnxA5 和 PS 配合物在微绒毛状的 membranesrepresent 中的核核 (NC) 在 M 的 Annexins 和 TNAP也具有胶原结合可能有助于在胶原纤维上放置 mv, 并在促进电解质中的矿化传播方面的能力。牛胎球蛋白 A 和骨桥蛋白 (OPN)¹⁷, 被称为磷灰石形成抑制剂, 可能减慢矿化在胶原支架上的传播。成核和传播是不同的事件, 前者前, 两者可能与病理成矿过程有关。

为了发现磷酸钙络合物的化学物质如何改变生理成矿作用和异位钙化, 有必要确定细胞产生的矿物质。磷灰石是一组含有矿物质的钙和磷酸盐, 一般晶体单元细胞配方钙₁₀(PO₄)₆X₂, 其中 X = Cl, F, OH。分类如下¹⁸: 氟磷灰石 (FA) 钙₁₀(PO₄)₆F₂, chlorapatite (ca) Ca₁₀(PO₄)₆Cl₂和羟基磷灰石 (HA) 钙₁₀(po₄)₆(OH)₂。

由于每一个细胞系都呈现出明显的矿化特征, 因此选择成骨细胞系诱导矿物形成是至关重要的。在这份报告中, 我们比较了两个选定的人体细胞模型的矿物成核: 成骨 hFOB 1.19 细胞和骨肉瘤 Saos-2 细胞。骨肉瘤衍生细胞通常被用作成骨模型, Saos-2 细胞保存了最成熟的成骨¹⁹ , 而未分化的人胎儿 hFOB 细胞作为正常成骨模型被广泛使用。分化²⁰。用不同的方法分析了它们的成矿剖面: 茜素红 s (AR) 染色、紫外 (紫外) 光可视化、透射电镜 (TEM) 成像、能量色散 X 射线微分析 (EDX) 定量和离子映射。EDX 在以往研究中使用的替代技术的优势在于, 它给出了⁴^、⁵^、²¹磷灰石晶体中离子置换的定量和定性结果。利用 TEM EDX 的总体目标是在成矿过程的不同阶段, 寻找一种简单的方法, 用于成像和定量的各种矿物中的钙、F 和 Cl 离子的分布。该方法成功地用于监测锌纳米粒子与共存化学物质的相互作用及其对水生生物的联合作用²²。在另一项研究中, 用电感耦合等离子体发射光谱 (ICP)、N2 储 (注)、XRD、紫外-比 DRS、红外光谱、拉曼光谱等方法, 对钛材料在水溶液中的铜光催化剂进行了广泛的表征。光谱学, 透射电镜 EDX 和光电测量²³。我们的目的是比较两个细胞系中的囊泡和矿物质的来源和性质, 以了解在骨分化过程中控制成矿作用的机制。

图 1.从膜中合成细胞外基质 (ECM) 蛋白和释放基质泡 (mv) 的骨细胞中初始化的步骤方案。通过钙结合蛋白、annexins 和磷酸酯的作用, 通过无机磷酸盐转运体 (坑) 的作用, 通过组织非特异碱性磷酸酶 (TNAP) 的活性来积累钙, dephosphorylatesPP_i对 P_i, 从而促进磷灰石成核。然后, 磷灰石分解并释放出细胞外培养基。矿化由 P_i和 Ca^{2 +}在胞外介质⁴^,⁵的恒定供应维持。请单击此处查看此图的较大版本.

Protocol

1. 细胞培养和治疗将所有必要的材料放在层流罩下, 并在紫外线照射下消毒。文化媒介是: 1:1 混合火腿的 F12 和 DMEM 培养基与2.5 毫米 l-谷氨酰胺补充100的 u/毫升青霉素, 100 U/毫升链霉素, 0.3 毫克/毫升 G418 和10% 胎牛血清 (v/v) 为人类胎儿 hFOB 1.19 SV40 大 T 抗原转染成骨细胞, 和麦考伊的5A 培养基与1.5 毫米 l-谷氨酰胺补充 100 u/毫升青霉素, 100 u/毫升链霉素和15% 血清 (v/v) 的人骨肉瘤 Saos-2 细胞。</…

Representative Results

EDX 允许在体外成像的基质囊泡 (mv) 释放的矿化细胞和矿物生产的 MVs.利用该技术得到的结果表明, 矿化过程可能会有不同的进展。在不同类型的细胞中。两个细胞系接受相同的成骨细胞转分化治疗, 但刺激 Saos-2 单元矿化更有效地比 hFOB 1.19 成骨细胞, 由 AR 染色证明 (图 2)。这可能是由于更成熟的成骨细胞表型的 Saos-2 单元19</…

Discussion

在本文中, 我们描述了 AR 染色的协议, 氟磷灰石的紫外光识别和透射电镜-EDX 的体外成像中的矿化细胞和磁气产生的矿物质.通过遵循一些常见的故障排除步骤, 可以解决上述所有方法。为了获得最佳结果, 应该仔细地执行几个关键步骤。首先, 最好添加 AA (酸性), 其次是β GP (碱性), 以保持培养基的 pH 值7.4。其次, 经过 AR 染色后, 染色的钙沉积物非常脆弱, 细胞应小心清洗, 以防止在添加 …

Disclosures

The authors have nothing to disclose.

Acknowledgements

MK 和要求执行手工操作和 LB 准备的图纸和制作的电影。问写的手稿, LB 写的剧本和 MK 准备的表。SM、RB 和 SP 认真阅读了表、剧本和手稿。作者感谢汉娜 Chomontowska 为 ultramicrotomy 以及叔父 Suski 和亨瑞 Bilski 提供了出色的协助, 他们对 TEM EDX 分析提供了出色的帮助。作者要感谢帕特里克林博士的专业英语语言更正和芭芭拉 Sobiak 记录的指示。

这项工作得到波兰科学和高等教育部赠款 N N401 140639 的支持, 由国家科学中心提供赠款, 波兰 2016/23/n/NZ4/03313 至磅和 2016/23/n/NZ1/02449 至 MK, 欧盟 FP7 项目 BIOIMAGINE: 生物成像在研究创新和教育, GA 264173, 和由 Nencki 实验生物学研究所的法定资金, 波兰科学院。

Materials

Reagent
Ham’s DMEM/F12 media mixture	PAA	E15-813	1:1, for human fetus hFOB 1.19 SV40 large T antigen transfected osteoblasts (ATCC CRL-11372)
McCoy’s 5A medium	PAA	E82312-0025	for human osteosarcoma Saos-2 cells (ATCC HTB-85)
Antibiotics mixture (penicillin/streptomycin)	Sigma	P0781-100ML	100 U/mL each
G-418	Sigma	68168	0.3 mg/mL
FBS	Gibco	10270	10% for hFOB 1.19 and 15% for Saos-2
AA	Sigma	A-5960	50 µg/mL
ß-GP	Sigma	G9422-100G	7.5 mM
Bio-Gel HTP Gel	Bio-Rad	130-0420	for HA
FA			synthesized by us
CA			synthesized by us
Sodium phosphate buffer Na₂HPO₄/NaH₂PO₄ mixture	Sigma	S7907/S8282	0.1 M, pH 7.2
PBS			pH 7.0, prepared by us
AR-S in PBS	Sigma	A5533-25G	0.5 g/100 mL, pH 5.0
Collagenase type IA	Sigma	C2674	500 U/mL
SCL buffer			prepared by us
Deionized wather			produced by us
Ethanol	POCh	BA6480111	absolut 99.8% and solutions 25, 50, 75, 90%
Uranyl acetate in 50% ethanol	Polysciences Inc.	21447-25	0.25 g/10 mL
PD medium			pH 7.4, prepared by us
Fixation mixture (paraformaldehyde/glutaraldehyde)	Sigma	158127/G-6257	3%:1%
Post-fixation OsO₄	Sigma	75633	1%
LR White resin in ethanol	Polysciences Inc.	17411-MUNC 500g	1:2, 1:1, 100%
Acetone	CHEMPUR	111024800	pure
Tool
Cryogenic vials	Corning Inc.	430487	1.2 mL
Plastic Petri culture dishes	Falcon	353003	100 mm
Plastic tubes	Falcon	352096 and 352070	15 and 50 mL
Serological pipettes	Falcon and VWR	357521 and 612-3700	1 and 10 mL
Plastic microcentrifuge tubes	Sigma	Z688312 and Z628034	1.5 mL black and 2 mL transparent
Plastic tips	VWR	613-0364, 613-0239 and 613-1050	0.1-10 µL natural, 1-200 µL yellow and 200-1000 µL blue
Plastic racks	Light Labs	A-7055-Z, A-7053-C	green for tubes, orange for micro tubes and blue for TEM probes
Laminar Hera Save	Thermo Scientific Co.	KS12	HEPA filter (H14 according to DIN EN 1822)
Incubators Hera Cell	Thermo Scientific Co.	150	34°C for hFOB 1.19 and 37°C for Saos-2
Fume hood	POLON	WCS-2	for TEM stainings
Glass bottles	SIMAX	1632414501050 and 1632414501100	50 and 100 mL
Quartz glass tubes	SIMAX	638422010100	Ø 10 mm, L 100 mm
Pump	IBS Integra Biosciences	VACUSAFE comfort	for vacuum
Oven	Memmert	UNE 400	56°C
Porcelain multi-well plate	Rosenthal technik	229/12	12 wells
Glass beakers	SIMAX	632417010025	25 mL
Glass bottles	Pocord	DIN22	10 mL
Plastic box	Agar Scientific Ltd.		for darkness
Snap Fit Gelatin Capsules	Agar Scientific Ltd.	G3741	size 1
Formvar/Carbon 300 Mesh Ni grids in box	Agar Scientific Ltd.	S162N3	film on the shiny side
Silicon cell scraper	Sigma	SIAL0010-100EA	size 1.8/25 cm
Syringe with needle	BogMark	007	syringe 1 mL 40 U, needle 0.5 x 16
Syringe	Chirana	CH005L	5 mL
Centrifuge	MPW Medical Instruments	MPW-350R	130 x g and 500 x g
UV transluminator	UVP	M-20	for visible and UV light
Ultramicrotome	LKB	NOVA	700Å sections
Block holder	LKB	E6711	round shape
Diamond knife	DiATOME	Ultra 45°	size 3
Eyelash holder			bovine, prepared by us
Forceps	ROTH	2855.1	antistatic for grids
Spatulas set	ROTH	E286.1	antistatic for powders
Imaging
Inverted Light Microscope	Zeiss with Canon	AxioObserver Z1 equipped with PowerShot G9	Phase contrast, Transmitted light, 20 x objective, RGB filters
Transmission Electron Microscope	TEM Jeol Co. with Oxford Instruments and SiS-Olympus	JEM-1400 TEM equipped with full range INCA Energy Dispersive X-ray microanalysis (EDX) System and 11 Megapixel MORADA G2 camera	magnification 50,000X for TEM and 15,000X for STEM and EDX
Camera body and lenses	Nikon	Nikon D7100 Nikkor AF Micro 105 mm f/2.8D Nikkor AF-S 50 mm f/1.8G Nikkor AF 28 mm f/2.8D	for movie recordings
Microphone	MXL Mics	Tempo	for voice recordings

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Bozycki, L., Komiazyk, M., Mebarek, S., Buchet, R., Pikula, S., Strzelecka-Kiliszek, A. Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis. J. Vis. Exp. (136), e57423, doi:10.3791/57423 (2018).