成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型
Summary
在这里,我们提出了一个成人小鼠端程phalanx截肢的协议,以研究哺乳动物的气肿形成和膜内性渗透,通过荧光免疫组织化学和连续体内微计算断层扫描进行分析。
Abstract
在这里,我们提出了成人小鼠远端咽喉(P3)截肢的协议,一个程序简单和可重复的哺乳动物表皮再生模型,其中涉及气肿形成和膜内性内性性荧光免疫组织化学和连续体内微计算断层扫描(μCT)。哺乳动物再生仅限于截肢,横断端法兰克斯(P3) 的远端区域;在更接近的水平上被截肢的数字不能再生,并经历纤维化愈合和疤痕形成。再生反应由增殖性爆发性气肿的调解,然后通过宫内骨质再生来恢复被截肢的骨骼长度。P3截肢是研究哺乳动物表皮再生的临床前模型,是设计治疗策略的有力工具,用成功的再生反应取代纤维化愈合。我们的方案使用荧光免疫组织化学 1) 识别早期和晚期的囊肿细胞群,2) 在再生环境中研究再血管化,3) 无需复杂骨骼即可研究膜内渗透稳定装置。我们还演示了使用连续体在体内μCT来创建高分辨率图像来检查截肢后的形态变化,以及量化再生过程中相同数字的体积和长度变化。我们相信,该协议为研究哺乳动物的表型和组织再生反应提供了巨大的效用。
Introduction
哺乳动物,包括人类和小鼠,在端法兰克斯(P3)1,2,3的远端截肢后,有能力再生其数字的尖端。在小鼠中,再生反应是截肢水平依赖;越来越近的数字截肢显示逐渐衰减的再生反应,直到完全再生失败在截肢横断和近端的P3指甲矩阵4,5,6,7,8.P3再生由形成大爆炸体介导,定义为通过形态形成来再生被截肢结构的增殖细胞群9。形成大爆炸体以再生截肢失去的结构,这个过程称为表皮再生,将多组织级P3再生反应与损伤后的传统组织修复区分开来。 10.P3再生是一个可重复和程序简单的模型,研究复杂的再生过程,包括伤口愈合11,12,骨质变11,12,再血管化13,外周神经再生14,并通过宫内骨化15向骨转化。
以前使用免疫性化学的研究表明,气肿是异质的,血管,缺氧,和高度增殖11,13,15,16。在远端P3截肢后,早期大发肿最初与P3型骨质和内膜有关,其特征是骨表面15附近有旺盛的增殖和新生的成骨。在骨降解和伤口闭合之后,异质性大爆发肿由围锁细胞和内窥镜相关细胞的合并形成,然后通过膜内骨化分化包括骨骼在内的囊肿成分分化15.
骨修复对损伤的反应通常发生内肠骨化,即通过初始的软骨,形成一个模板,为后续的骨形成17,18 。长骨内膜化,即骨形成无软骨中间体,通常诱导使用复杂的分心装置或手术固定19,20。数字再生反应是一种临床前模型,与传统的膜内沉降模型相比具有优势:1) 它不需要外部或内部固定损伤后刺激膜内沉降,2)使用每只动物的 4 位数字进行,从而在最大程度地减少动物使用的同时最大化样品,并且 3) 可轻松、快速地执行体内连续微计算断层扫描 (μCT) 分析。
在本研究中,我们展示了标准化的P3截肢平面,以实现可重复和强健的再生反应。此外,我们演示了优化的荧光免疫组织化学方案,使用石蜡部分来可视化气肿形成、再生环境中的再血管化,以及通过膜内向骨的气管转换骨化。我们还演示了在再生过程中使用连续体内-CT来识别同一数字的骨骼形态、体积和长度的变化。该协议的目的是研究截肢后的哺乳动物气肿形成,并演示2种技术,荧光免疫组织化学和连续体内-CT,用于研究宫内骨再生。
Protocol
Representative Results
Discussion
该协议描述了成人小鼠远端P3截肢的标准化程序,荧光免疫组织化学染色,以可视化和调查气肿形成和膜内性渗透,并连续在体内进行_CT扫描到识别截肢后的骨形态、体积和长度变化。P3截肢是一种独特的,程序简单,可重复的模型,以分析一个促进再生的伤口环境,触发出血形成。此外,P3数字模型具有许多优点,比传统的骨损伤模型,以研究膜内骨化。
为确保此协议的成功,必须执行完整…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢穆尼奥实验室和得克萨斯基因组医学研究所(TIGM)的成员。这项工作得到了德克萨斯A&M大学的支持。
Materials
| Protein Block Serum Free | DAKO | X0909 | Ready to use |
| Mouse anti-PCNA antibody | Abcam | ab29 | 1:2000 dilution |
| Rat anti-CXCR4 antibody | R&D Systems | MAB21651 | 1:500 dilution |
| Rabbit anti-human vWF XIII antibody | DAKO | A0082 | 1:800 dilution |
| Rabbit anti-osterix, SP7 antibody | Abcam | ab22552 | 1:400 dilution |
| Rabbit anti-Runx2 antibody | Sigma-Aldrich Co. | HPA022040 | 1:250 dilution |
| Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L) | Invitrogen | A21235 | 1:500 dilution |
| Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) | Invitrogen | A11008 | 1:500 dilution |
| Alexa Fluor 568-conjugated goat anti-rat IgG (H+L) | Invitrogen | A11077 | 1:500 dilution |
| Prolong Gold antifade reagent | Invitrogen | P36930 | Ready to use |
| Surgipath Decalicifier 1 | Leica Biosystems | 3800400 | Ready to use |
| Z-Fix, Aqueous buffered zinc formalin fixative | Anatech LTD | 174 | Ready to use |
| CD-1 Female Mouse | Envigo | ICR(CD-1) | 8-12-weeks-old |
| vivaCT 40 | SCANCO Medical |
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