在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪
Summary
该协议描述了一种有效的方法来监测人脂肪来源的骨髓间充质干细胞 (haMSCs) 的细胞持久性和分布的远红色荧光标记的大鼠膝骨关节炎 (KOA) 模型通过关节内 (IA) 注射。
Abstract
为了支持人脂肪源性间充质干细胞 (haMSC) 治疗膝骨关节炎 (KOA) 的临床应用, 我们研究了 haMSCs 在动物模型中的细胞持久性和分布的有效性。我们展示了一种用亲油性荧光染料标记 haMSCs 细胞膜的方法。随后, 由体内成像系统动态监测经手术诱导的 KOA 大鼠的关节内注射标记细胞。我们雇用的亲油性 carbocyanines 做了 (DilC18 (5)), 一个远红色荧光 Dil (dialkylcarbocyanines) 模拟, 它利用红色激光, 以避免激发自然绿色自发荧光从周围的组织。此外, 红移发射光谱的确有允许深层成像在活体动物和标记程序没有造成细胞毒作用或功能损害 haMSCs。这种方法已被证明是一种有效的跟踪方法的 haMSCs 在大鼠 KOA 模型。该方法的应用也可用于确定临床前研究中其他来源的骨髓间充质干细胞的最佳用药路线和剂量。
Introduction
膝关节骨关节炎 (KOA) 是一种退行性疾病, 由关节软骨丧失和渐进炎症, 这已成为一个主要的慢性病在世界各地的老年人1。然而, 目前的治疗使用抗炎药物, 物理补充剂和手术程序可能只提供暂时缓解症状性疼痛2。
人脂肪源性间充质干细胞 (haMSCs) 已成为一种有希望的再生治疗膝关节骨性关节炎, 由于其多能分化潜能的软骨再生和免疫调节特性3, 4。与药理学途径, 以研究的作用机制的行为在体内, 跟踪活 haMSCs 在小 KOA 动物模型目前是有益的建立的理由和可行性 haMSC 治疗前的临床应用。在临床前测试中, 内侧切除 (MM) 破坏关节的机械负荷诱导大鼠 KOA, 提供了一个相对可行的模型, 具有一致的重现性5。KOA 诱发的早于前十字韧带横断, 或与部分内侧切除6相结合。因此, 在由 MM7、8引起的大鼠中, 通常会对注射 haMSCs 与 KOA 的病理微环境之间的长期相互作用进行评估。
虽然 haMSCs 的治疗效果已被广泛报道, 有关知识的体内持续的植入 haMSCs 通过关节内 (IA) 注射是稀缺的9,10。因此, 已经开发了各种细胞标记方法, 包括 immunohistology11、荧光12、绿色荧光蛋白13转染、氧化铁标记为磁共振成像 (MRI)14和众多荧光细胞染料8、15、16。与 labor-intensive 组织学分析相比,在体内非侵入性成像采用光学器件来检测标记有荧光信号的细胞的 real-time 分布和动态,1017。对于功能性活细胞成像, cytocompatible 荧光标记是一种先进的无放射性跟踪技术, 以揭示细胞活动后, 干细胞移植18。此外, 多色荧光亲油性染料具有相对于氨基活性亲水染料或荧光蛋白的优势, 包括其改进的细胞通透性和增强的荧光量子产率19。
因此, 这里所包含的协议使用红色激光激发带有亲油性 carbocyanines 的细胞 (DilC18(5)), 这是一个远红色荧光 Dil (dialkylcarbocyanines) 模拟20。红移激发和发射光谱的确有避免 autofluorescent 干扰, 并允许深层成像在很长一段时间的活动物8。这种跟踪细胞在体内的方法是有效的监测移植的干细胞, 如 haMSCs, 在动物模型, 这是必不可少的了解和改善现有的干细胞再生疗法。
Protocol
Representative Results
Discussion
我们迫切需要干细胞治疗的安全标准和分布研究, 才能将再生干细胞治疗 KOA 从长凳到床边。然而, 疾病的病理环境在移植 haMSCs 的持续和分布中起着重要作用10。最近, 我们的小组表明, 关节内注射 haMSCs 在病理 KOA 环境中的持续时间比正常情况下注射在8。炎症和退行性微环境可能会招募骨髓间充质干细胞进行修复, 随后有利于保留注入的 haMSCs1<s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由上海市科技委员会 (CN) 赞助的上海创新基金 (1402H294300) 资助, Dr.。感谢 Dr. 广东周 (中国国家组织工程中心) 对本论文的技术支持和科学建议。我们还要感谢 Mr. 矿泉夏 (上海第九人民医院) 在动物福利方面的帮助。
Materials
| Matrx VMR animal anesthesia system | Midmark | VIP3000 | |
| 4-0 suture | Shanghai Jinhuan | KC439 | |
| Razor | Pritech | LD-9987 | |
| Gentamicin | Zhejiang Jindakang Animal Health Product Co., Ltd. | None | |
| 0.9% Sodium chloride solution | Hunan Kelun Pharmaceutical Co., Ltd. | H43020455 | |
| Penicillin | Shanghai Kangfu chemical pharmaceutical Co., Ltd. | None | |
| Buprenorphine | Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd. | None | |
| Paraformaldehyde | Sigma-Aldrich | 16005 | Dilute to final concentration of 10% in PBS |
| EDTA | Sigma-Aldrich | E9884 | Dilute to final concentration of 20% in PBS |
| 0.1% Hematoxylin Solution, Mayer’s | Sigma-Aldrich | MHS16 | |
| 0.5% Eosin Y solution, alcoholic | Sigma-Aldrich | HT110116 | |
| Safranin O | Sigma-Aldrich | S8884 | |
| Fast Green | Sigma-Aldrich | F7258 | |
| Shandon Excelsior ESTM Tissue Processor | Thermo Fisher | A78400006 | |
| Shandon Histocentre™ 3 Tissue Embedding Center | Thermo Fisher | B64100010 | |
| Fully Automated Rotary Microtome | Leica | RM2255 | |
| DiD | Molecular Probes, Life Technologies |
V-22887 | |
| D-MEM High Glucose | Sigma-Aldrich | D5648 | |
| PBS | GIBCO, Life Technologies | 14190-144 | |
| 0.25% Trypsin-EDTA | Invitrogen | 25200-114 | |
| 10 cm Petri Dish | Corning | V118877 | |
| Centrifuge | Beckman | Optima MAX-TL | |
| Fluorescent microscope | Olympus | BX53 | |
| 0.4% Trypan Blue solution | Sigma-Aldrich | 93595 | |
| Titetamme | Virbac (Zoletil 50) | 1000000188 | |
| Zolazepam | Virbac (Zoletil 50) | 1000000188 | |
| Sterile hyposermic syringe for single use 26G | Shanghai Misawa Medical Industry | None | |
| IVIS Spectrum In Vivo Imaging System | PerkinElmer | 124262 | |
| Living Imaging 4.0 software | PerkinElmer | None |
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