脐带间充质干细胞外泌体修复骨质疏松大鼠颅骨缺损的研究

Exosomes derived from umbilical cord mesenchymal stem cells repair skull defect of osteoporosis rats: an experimental study

摘要:

目的： 探讨人脐带间充质干细胞（human umbilical cord mesenchymal stem cells，HucMSCs）源外泌体对骨质疏松大鼠颅骨缺损的修复作用。方法： 提取HucMSCs来源外泌体，使用透射电镜及纳米颗粒追踪分析鉴定其形态及大小，使用Western blot鉴定其表面标志物；分离培养大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）并使用流式细胞术鉴定其表面标志物；将外泌体（空白对照、20 μg/mL、40 μg/mL、60 μg/mL、80 μg/mL）与BMSCs共培养，检测不同浓度的外泌体对BMSCs增殖及成骨向分化的影响；选取SD大鼠通过卵巢切除术（ovariectomy，OVX）构建骨质疏松模型，在颅骨缺损模型建立的同时注射外泌体悬液，术后8周处死大鼠，取术区组织进行micro-CT扫描并进行骨量分析，HE染色后显微镜下观察骨缺损愈合情况，评价HucMSCs来源外泌体对骨质疏松大鼠颅骨缺损的修复潜能。结果： 透射电镜及纳米颗粒追踪分析结果显示，提取的HucMSCs来源外泌体形态及大小符合要求，表面蛋白标志物CD63及CD81表达均呈阳性。大鼠BMSCs表面标志物CD29高表达（99.86%）、CD90高表达（99.50%），CD45低表达（4.12%），符合间充质干细胞特征。将60 μg/mL的外泌体与BMSCs共培养5 d后细胞活力为对照组的（127.782±7.184）%（P=0.001），碱性磷酸酶（alkaline phosphatase，ALP）的表达为对照组的（2.715±0.095）倍（P=0.003），Runx家族转录因子2（Runx family transcription factor 2，Runx2）的表达为对照组的（2.175±0.731）倍（P=0.140）。在OVX诱导的骨质疏松SD大鼠颅骨缺损模型的修复中，micro-CT显示外泌体组相对于对照组出现了明显的骨愈合增加，骨量分析结果显示外泌体组骨组织比例为（11.000±1.708）%（P=0.008），骨小梁数目为（0.742±0.112）mm^-1（P=0.014），骨小梁厚度为（0.175±0.008）mm（P=0.005），骨小梁间隙为（1.402±0.507）mm（P=0.022）。组织学结果显示新生骨的结构完整、层次清晰，为致密的板层样骨，有均匀的骨基质和骨陷窝。结论： HucMSCs来源的外泌体能够促进BMSCs的增殖活性及成骨向分化，具有修复骨质疏松骨缺损的能力。

Abstract:

Objective: To explore the repairing effect of exosomes derived from human umbilical cord mesenchymal stem cells (HucMSCs) on the skull defect of osteoporosis rats. Methods: Isolation the exosomes from HucMSCs, the morphology and size of the exosomes were identified by transmission electron microscopy and nanoparticle tracking analysis, and the surface markers were identified by Western blot. Rat bone marrow mesenchymal stem cells (BMSCs) were isolated and their surface markers were identified by flow cytometry. Exosomes (control, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL) were co-cultured with BMSCs, and the effects of different concentra-tions of exosomes on proliferation and early osteogenic differentiation of BMSCs were detected. Osteoporosis model was established by ovariectomy (OVX) in SD rats. Exosomes suspension was injected at the same time as the skull defect model was estab-lished. Eight weeks after operation, the rats were euthanized. micro-CT scanning and bone mass analysis was performed on the tissue of the operation area, and the healing of bone defect was observed under microscope after HE staining, so as to evaluate the repairing potential of exosomes from HucMSCs on the skull defect of osteoporosis rats. Results: The results of transmission electron microscope and nanoparticle tracking analysis showed that the shape and size of exosomes derived from HucMSCs met the requirements, and the expression of surface protein markers CD63 and CD81 were positive. The surface markers CD29, CD90 and CD45 of rats BMSCs were expressed as 99.86%, 99.50% and 4.12% respectively, which accorded with the characteristics of mesenchymal stem cells. After co-culturing 60 μg/mL exosomes with BMSCs for 5 days, the cell viability was (127.782±7.184) % of the control group (P=0.001), the expression of alkaline phosphatase (ALP) was (2.715±0.095) times of the control group (P=0.003), and the expression of Runx family transcription factor 2 (Runx2) was (2.175±0.731) times of the control group (P=0.140). In the repair of an OVX-induced osteoporosis SD rat skull defect model, micro-CT showed that the exosome group had a significant increase in bone healing compared with the control group. The results of bone mass analysis showed that in the exosome group, the bone volume/tissue volume was (11.000±1.708) % (P=0.008), the trabecular number was (0.742±0.112) mm^-1 (P=0.014), the trabecular thickness was (0.175±0.008) mm (P=0.005), and the trabecular separation was (1.402±0.507) mm (P=0.022). Histological results showed that the new bone had a complete structure, with clear layers, as dense lamellar bone, and uniform bone matrix and bone lacunae. Conclusion: The exosomes derived from HucMSCs can promote the proliferation and osteogenic differentiation of BMSCs, and have the ability to repair osteoporotic bone defects.