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Serum-derived exosomes from neurofibromatosis type 1 congenital tibial pseudarthrosis impaired bone by promoting osteoclastogenesis and inhibiting osteogenesis
Experimental Biology and Medicine ( IF 2.8 ) Pub Date : 2020-10-06 , DOI: 10.1177/1535370220962737
Ge Yang 1 , Hui Yu 2 , Yaoxi Liu 1 , Weihua Ye 1 , Guanghui Zhu 1 , An Yan 1 , Qian Tan 1 , Haibo Mei 1
Affiliation  

Treatment of congenital pseudarthrosis of the tibia (CPT) still is full of challenges in pediatric orthopedist. Serum-derived exosomes (SDEs) have been proven to be participated in bone remodeling. However, the molecular changes in SDEs of CPT children and their pathologies have not been elucidated. In this study, SDEs were isolated and purified from CPT patients (CPT-SDEs) associated with neurofibromatosis type 1 (NF1) and normal children (Norm-SDEs). Then we obtained the proteomics profile of SDEs by combining liquid chromatography-tandem mass spectrometry (LC-MS/MS) and tandem mass tag label-based quantitation. In vitro, the efficacy of SDEs on osteoblastic differentiation of MC3T3-E1 cells and osteoclastogenesis ability of RAW264.7 cells were evaluated by quantitative real-time PCR (qRT-PCR) and cytochemical staining. In vivo, we used micro-CT to assess cortical bone mass and trabecular microstructures to reflect the influence of SDEs on bone remodeling after injection into the tail vein of rats. Based on proteomics analysis, 410 differentially expressed proteins, including 289 downregulated proteins and 121 upregulated proteins, were identified in the CPT-SDEs. These proteins have multiple biological functions associated with cellular metabolic processes, catalytic activity, and protein binding, which are important for cell differentiation and proliferation. In vitro, CPT-SDEs decreased the osteogenic differentiation of MC3T3-E1 cells and promoted the osteoclastogenesis of RAW264.7 cells. Injection of CPT-SDEs into the tail vein for two months resulted in bone loss in rats, as indicated by the decrease in trabecular and cortical bone mass. Our findings demonstrated the differences in proteins in SDEs between normal and CPT children with NF1. These differentially expressed proteins in CPT-SDEs contributed to deteriorating trabecular bone microstructures by inhibiting bone formation and stimulating bone resorption.

Impact statement

Congenital pseudarthrosis of the tibia (CPT) is an uncommon and puzzling disease associated with a high rate of disability. To date, the biological mechanisms related to this disease have largely not been elucidated. In this study, we determined the biological functions of serum-derived exosomes (SDEs) from children with neurofibromatosis type 1 (NF1) associated with CPT (CPT-SDEs) and compared their proteomic profiles with those of SDEs from normal children. Based on proteomics analysis, 410 differentially expressed proteins, including 289 downregulated proteins and 121 upregulated proteins, were identified in the CPT-SDEs. In addition, CPT-SDEs decreased the osteogenic differentiation of MC3T3-E1 cells and promoted the osteoclastogenesis ability of RAW264.7 cells. Moreover, injecting CPT-SDEs into the tail veins led to bone loss in rats, as detected by the reduction in trabecular and cortical bone mass. These findings indicate that CPT-SDEs impair bone quality, which may provide a reasonable explanation for the low bone quality and tibial nonunion in children with NF1 associated with congenital tibial pseudarthrosis.



中文翻译:


来自 1 型神经纤维瘤病先天性胫骨假关节的血清来源的外泌体通过促进破骨细胞生成和抑制成骨而损害骨



先天性胫骨假关节(CPT)的治疗对儿科骨科医生来说仍然充满挑战。血清源性外泌体(SDE)已被证明参与骨重塑。然而,CPT儿童SDE的分子变化及其病理学尚未阐明。在这项研究中,从与 1 型神经纤维瘤病 (NF1) 相关的 CPT 患者 (CPT-SDE) 和正常儿童 (Norm-SDE) 中分离和纯化了 SDE。然后,我们通过结合液相色谱-串联质谱 (LC-MS/MS) 和基于串联质量标签标记的定量,获得了 SDE 的蛋白质组学特征。在体外,通过实时定量PCR(qRT-PCR)和细胞化学染色评估SDE对MC3T3-E1细胞成骨细胞分化和RAW264.7细胞破骨细胞生成能力的功效。在体内,我们使用显微CT评估皮质骨量和小梁微结构,以反映SDE注射到大鼠尾静脉后对骨重塑的影响。基于蛋白质组学分析,在CPT-SDE中鉴定出410个差异表达蛋白,其中包括289个下调蛋白和121个上调蛋白。这些蛋白质具有与细胞代谢过程、催化活性和蛋白质结合相关的多种生物功能,这对于细胞分化和增殖很重要。在体外,CPT-SDEs 降低了 MC3T3-E1 细胞的成骨分化,促进了 RAW264.7 细胞的破骨细胞生成。将 CPT-SDE 注射到尾静脉两个月会导致大鼠骨质流失,如小梁骨和皮质骨量减少所示。 我们的研究结果证明了患有 NF1 的正常儿童和 CPT 儿童 SDE 中蛋白质的差异。 CPT-SDE 中这些差异表达的蛋白质通过抑制骨形成和刺激骨吸收而导致骨小梁微结构恶化。

 影响报告


先天性胫骨假关节(CPT)是一种罕见且令人费解的疾病,与高致残率相关。迄今为止,与该疾病相关的生物学机制在很大程度上尚未阐明。在这项研究中,我们确定了与 CPT 相关的 1 型神经纤维瘤病 (NF1) 儿童的血清源性外泌体 (SDE) (CPT-SDE) 的生物学功能,并将其蛋白质组谱与正常儿童的 SDE 进行了比较。基于蛋白质组学分析,在CPT-SDE中鉴定出410个差异表达蛋白,其中包括289个下调蛋白和121个上调蛋白。此外,CPT-SDEs还降低了MC3T3-E1细胞的成骨分化能力,并促进了RAW264.7细胞的破骨细胞生成能力。此外,将 CPT-SDE 注射到尾静脉中会导致大鼠骨质流失,这可以通过小梁骨和皮质骨量的减少来检测。这些研究结果表明,CPT-SDE 会损害骨质量,这可能为先天性胫骨假关节相关NF1儿童的骨质量低下和胫骨骨不连提供合理的解释。

更新日期:2020-10-07
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