Summary

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

Published: September 16, 2020
doi:

Summary

该协议描述了使用活体动物活体显微镜实时检测CCL5介导的骨膜骨骼干细胞迁移。

Abstract

骨膜骨骼干细胞(P-SSC)对于终身骨骼维护和修复至关重要,使其成为开发增强骨折愈合的疗法的理想焦点。 骨膜细胞迅速迁移到损伤处,为骨折愈合提供新的软骨细胞和成骨细胞。传统上,细胞因子诱导细胞迁移的功效仅在体外通过进行转孔或划痕测定进行。随着使用多光子激发的活体显微镜检查的进步,最近发现1)P-SSC表达迁移基因CCR5和2)使用称为CCL5的CCR5配体处理改善了骨折愈合和P-SSC响应CCL5的迁移。这些结果是实时捕获的。这里描述的是一种方案,用于可视化P-SSC从颅骨架骨骼干细胞(SSC)生态位迁移到CCL5治疗后的损伤。该协议详细介绍了小鼠约束装置和成像支架的构造,小鼠颅骨的手术准备,颅骨缺陷的诱导以及延时成像的采集。

Introduction

骨折修复是一种动态的多细胞过程,与胚胎骨骼发育和重塑不同。在此过程中,从受损组织诱导损伤信号后,骨骼干/祖细胞的快速募集、增殖和随后分化,这些都对骨折的整体稳定性和固定至关重要1。特别是,骨折愈合的早期阶段需要软愈伤组织形成,这主要归因于骨膜常驻细胞2。当骨骼受伤时,骨膜细胞的一个亚群会迅速做出反应,并导致愈伤组织内新分化的软骨中间体和成骨细胞3,暗示骨膜内存在明显的骨骼干/祖细胞群。因此,骨膜驻留骨骼干细胞(SSC)的鉴定和功能表征是治疗退行性骨病和骨缺损的一种有前途的治疗方法4

SSC被认为存在于多个组织位置,包括骨髓。与骨髓类似,在骨膜中也发现了成骨/软骨SSC456。 这些骨膜 SSC (P-SSC) 可在胎儿骨骼发育过程中使用早期间充质谱系标志物(即 Prx1-Cre5Ctsk-Cre7Axin2-CreER8)进行标记478910这些单一遗传谱系追踪模型的一个显着局限性是标记细胞群中存在实质性的异质性。此外,它们无法将标记的SSC与其体内的后代区分开来。为了解决这一限制,我们最近开发了一种双报告小鼠(Mx1-Cre+ Rosa26-Tomato+α SMA-GFP+),以清晰地可视化骨髓SSC(BM-SSC)中的P-SSC11。通过该模型,已经确定P-SSC以Mx1 + αSMA +双重标记标记标记,而更多分化的Mx1 +细胞驻留在骨髓,骨内和骨膜表面以及皮质和小梁骨1112

已知多种细胞因子和生长因子可调节骨重塑和修复,并且已经过测试,以增强关键节段缺损中的骨骼修复113。然而,由于通过破坏骨室的物理屏障产生的损伤模型的细胞复杂性,这些分子对内源性P-SSC迁移和愈合期间激活的直接影响尚不清楚。SSC的功能特征和迁移动力学通常在体外通过进行转孔或划痕测定来评估,并结合已知可诱导其他细胞群迁移的细胞因子或生长因子。因此,解释这些体外实验的结果以应用于其相应的体内系统是具有挑战性的。目前,骨骼干/祖细胞迁移的体内评估通常不是实时观察到的;相反,它是在骨折后的固定时间点测量的57141516

这种方法的局限性是没有在单细胞水平上评估迁移;相反,它是通过细胞群的变化来测量的。由于活体动物活体显微镜检查和额外报告小鼠的产生的最新进展,现在可以对单个细胞进行体内跟踪。使用活体动物活体显微镜,我们观察到Mx1 / Tomato / αSMA-GFP小鼠在受伤后24-48小时内P-SSC从颅骨缝合龛位到骨损伤的不同迁移。

CCL5/CCR5最近被确定为一种调节机制,在早期损伤反应期间影响P-SSC的招募和激活。有趣的是,没有检测到大量的P-SSC迁移以响应实时损伤。然而,用CCL5治疗损伤会产生P-SSC的稳健,方向上不同的迁移,可以实时捕获。因此,该方案的目的是提供一种详细的方法,用于在用CCL5处理后实时记录P-SSC的体内迁移。

Protocol

所有小鼠都保持在无病原体的条件下,所有程序均由贝勒医学院的机构动物护理和使用委员会(IACUC)批准。 1. 鼠标准备 将Mx1-Cre17和Rosa26-loxP-stop-loxP-tdTomato(Tom)18只小鼠(从杰克逊实验室购买)与αSMA-GFP小鼠(由Ivo Kalajzic博士和Henry Kronenberg博士提供)杂交,生成Mx1-Cre + Rosa26-Tomato +</sup…

Representative Results

骨骼祖细胞被认为具有迁移或循环潜力20。最近,生成了Mx1-Cre+ Rosa26-Tomato+α SMA-GFP+(Mx1/Tomato/αSMA-GFP)报告小鼠,其中P-SSC用Mx1 + α SMA +双重标记标记(图2A,B)。Mx1+ α SMA+ P-SSC在损伤后24-48小时内从缝合间充质中大量迁移<sup cl…

Discussion

在骨愈合过程中,骨膜细胞是损伤愈伤组织内新分化的软骨细胞和成骨细胞的主要来源3。与骨髓类似,在骨膜中也发现了成骨/软骨SSC456。评估内源性P-SSC功能特征在技术上具有挑战性。通常,SSC的迁移动力学是在体外评估的,这使得对其相应体内系统的解释具有挑战性。由于活体动物活体活体显微镜检?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了德克萨斯州骨病计划奖,Caroline Wiess法律基金奖和美国国立卫生研究院NIAMS的支持,奖项编号为R01 AR072018,R21 AG064345,R01 CA221946至D.P。 我们感谢M.E. Dickinson和T.J. Vadakkan在BCM Optical Imaging and Vital Microscopy Core 以及BCM Advanced Technology Cores资助下,由NIH资助(AI036211,CA125123和DK056338)。

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Materials

½” optical post ThorLabs TR2 For imaging mount
1 mL syringe BD 309659
27G needle BD PercisionGlide 305111
29G insulin syringe McKesson 102-SN05C905P
50 mL conicol tube Falcon 352098 For mouse restraint
Adjustable angle plate Renishaw R-PCA-5023-50-20 For imaging mount
Alcohol wipes Coviden 6818
betadine surgical scrub Henry Schen 67618-151-16
Buprenorphine SR-LAB ZooPharm 1mg/mL Sustained Release
Combo III Obtained from staff veterinarian N/A 37.6 mg/mL Ketamine; 1.9 mg/mL Xylazine; 0.37 mg/mL Acepronazine
Coverslip Fisher 12-545-87 24 x 40 premium superslip
Fine tip forcepts FST 11254-20
Ketamine KetaVed 50989-161-06 100 mg/mL
Leica TCS SP8MP with DM6000CFS Leica Microsystems N/A
Matrigel R & D Systems 344500101
Medical tape McKesson 100199 3" x 10 yds (7.6 cm x 9.1 m)
Methocellulose Electron Microscopy Sciences 19560
Microdissection scissors FST 1456-12
Motorized stage Anaheim automation N/A
Needle holder FST 12500-12
Nonabsorbable sutures McKesson S913BX monofilament nylon 5-0 nonabsorbable sutures with attached C-1 reverse cutting needle
Opthalmic ointment Rugby 0536-1086-91
RANTES Biolegend 594202 10 µg/50 µL
Right-angle clamp for ½” post, 3/16” Hex ThorLabs RA90 For imaging mount
Spring-loaded 3/16” Hex-locking ¼” thumbscrew ThorLabs TS25H For imaging mount
Sterile cotton swabs Henry Schen 100-9249
Sterile DPBS (1x) Corning 21-030-CV
Sterile drapes McKessen 25-517
Surgical gloves McKessen 3158VA
Triple antibiotic ointment Taro Pharmaceuticals U.s.a., Inc. 51672-2120-2
Vacutainer blood collection set BD REF 367298 25G butterfly needle infusion set with 12" tubing

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Citazione di questo articolo
Ortinau, L., Lei, K., Jeong, Y., Park, D. Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice. J. Vis. Exp. (163), e61162, doi:10.3791/61162 (2020).

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