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| 骨髓间充质干细胞来源的外泌体对骨再生机制研究 |
| The mechanism of bone marrow mesenchymal stem cell-derived exosomes for bone regeneration |
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| DOI：10.3969/j.issn.1006-7108.2019.04.010 |
| 中文关键词: 外泌体 骨髓间充质干细胞 成骨分化 骨再生 |
| 英文关键词:exosomes bone marrow mesenchymal stem cells osteogenic differentiation bone regeneration |
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| 中文摘要: |
| 目的 本研究旨在检测骨髓间充质干细胞外泌体（BMSC-Exos）的特征，并通过体内体外实验探讨其对成骨分化的影响及对骨再生的机制。方法 ①原代培养人骨髓间充质干细胞（BMSCs），并对其表面抗原和多系分化潜能进行鉴定；②收集BMSC的P4~P6代细胞培养的培养上清液，应用试剂盒提取BMSC-Exos；③透射电镜观察BMSC-Exos的形态结构，免疫电泳检测BMSC-Exos的表面抗原；④茜素红、ALP染色验证BMSC-Exos在体外成骨分化中的作用；⑤通过大鼠颅骨缺损动物模型验证BMSC-Exos在体内骨再生的作用；⑥通过免疫电泳和qRT-PCR检测加入BMSC-Exos后的成骨细胞中相关蛋白和基因的表达情况。结果 ①分离培养的BMSCs形态呈多角形或长梭形，表面抗原CD90、CD29、CD44为阳性，符合间充质干细胞的特征；②BMSC-Exos呈双面凹的圆形或椭圆形，直径约40~120 nm（81.7±19.9），表面抗原与BMSC一致；③经茜素红染色和ALP染色后，染色强度与Exo浓度呈正相关；④通过对大鼠颅骨缺损模型拍摄X片、组织学分析，外泌体可促进大鼠颅骨缺损的修复和新生骨的形成；⑤免疫电泳显示经外泌体处理后，BMSC中的OCN、Runx2、β-catenin蛋白含量增加，qRT-PCR结果显示Runx2、β-catenin的基因表达上调。结论 BMSC-Exos有促进骨再生的能力，并与上调Wnt/β-catenin通路有关。 |
| 英文摘要: |
| Objective To investigate the characteristics of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) and to verify its ability to promote osteogenic differentiation through in vitro and in vivo experiments, as well as to explore its mechanism of promoting bone regeneration. Methods ① Primary human bone marrow mesenchymal stem cells (BMSCs) were cultured and their surface antigen and multilineage differentiation potentials were identified;② Culture supernatants of P4-P6 generation cell culture of BMSC were collected and passed. BMSC-Exos was extracted by the kit method; ③The morphological structure of BMSC-Exos was observed by transmission electron microscope; the surface antigen of BMSC-Exos was detected by immunoelectrophoresis; ④ Alizarin red and ALP staining was used to confirm that BMSC-Exos promoted osteogenic differentiation in vitro; ⑤ The ability of BMSC-Exos to promote bone regeneration was verified in vivo through a rat skull defect animal model;⑥The expression of related proteins and genes in osteoblasts after adding BMSC-Exos was detected by immunoelectrophoresis and qRT-PCR. Results ① The isolated and cultured BMSCs were polygonal or spindle-shaped, and the surface antigens CD 90, CD 29 and CD 44 were positive, which was consistent with the characteristics of mesenchymal stem cells; ② BMSC-Exos was of a biconcave round or oval shape with a diameter of about 40-120 nm (81.7±19.9), and the surface antigen was consistent with BMSC; ③ After Alizarin red staining and ALP staining, the Exo group staining intensity was significantly higher than that of the control group, and the staining intensity was positively correlated with the Exo concentration; ④X-ray and histological analysis of the rat skull defect model showed that the repair of the bone defect in the exosome group was better than that of the control group, and there were more new bone formation; ⑤Immunoelectrophoresis showed that after treatment with exosomes, the content of OCN, Runx 2 and β-catenin protein in MSC cells increased, and qRT-PCR showed that the gene expression of Runx2 and β-catenin was up-regulated. Conclusion: BMSC-Exos has the ability to promote bone regeneration and is associated with the up-regulation of Wnt/β-catenin pathway. |
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