沉默 p75NTR 可调节 BMSCs 的成骨分化和血管生成，从而增强骨折大鼠的骨愈合。

Silencing p75NTR regulates osteogenic differentiation and angiogenesis of BMSCs to enhance bone healing in fractured rats.

机构信息

Department of Trauma and Arthrology, First Affiliated Hospital of Shaoyang University, Shaoyang, Hunan, China.

出版信息

J Orthop Surg Res. 2024 Mar 20;19(1):192. doi: 10.1186/s13018-024-04653-8.

DOI:10.1186/s13018-024-04653-8

PMID:38504358

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10953090/

Abstract

BACKGROUND

Fractures heal through a process that involves angiogenesis and osteogenesis but may also lead to non-union or delayed healing. Bone marrow mesenchymal stem cells (BMSCs) have been reported to play a pivotal role in bone formation and vascular regeneration and the p75 neurotrophin receptor (p75NTR) as being an important regulator of osteogenesis. Herein, we aim to determine the potential mediation of BMSCs by p75NTR in bone healing.

METHODS

Rat BMSCs were identified by flow cytometry (FCM) to detect cell cycle and surface markers. Then transfection of si/oe-p75NTR was performed in BMSCs, followed by Alizarin red staining to detect osteogenic differentiation of cells, immunofluorescence double staining was performed to detect the expression of p75NTR and sortilin, co-immunoprecipitation (CO-IP) was conducted to analyze the interaction between p75NTR and sortilin, and EdU staining and cell scratch assay to assess the proliferation and migration of human umbilical vein endothelial cells (HUVECs). The expression of HIF-1α, VEGF, and apoptosis-related proteins were also detected. In addition, a rat fracture healing model was constructed, and BMSCs-si-p75NTR were injected, following which the fracture condition was observed using micro-CT imaging, and the expression of platelet/endothelial cell adhesion molecule-1 (CD31) was assessed.

RESULTS

The results showed that BMSCs were successfully isolated, p75NTR inhibited apoptosis and the osteogenic differentiation of BMSCs, while si-p75NTR led to a decrease in sortilin expression in BMSCs, increased proliferation and migration in HUVECs, and upregulation of HIF-1α and VEGF expression. In addition, an interaction was observed between p75NTR and sortilin. The knockdown of p75NTR was found to reduce the severity of fracture in rats and increase the expression of CD31 and osteogenesis-related proteins.

CONCLUSION

Silencing p75NTR effectively modulates BMSCs to promote osteogenic differentiation and angiogenesis, offering a novel perspective for improving fracture healing.

摘要

背景

骨折的愈合过程涉及血管生成和骨生成，但也可能导致骨不连或愈合延迟。骨髓间充质干细胞（BMSCs）在骨形成和血管再生中起着关键作用，而 p75 神经营养素受体（p75NTR）是骨生成的重要调节因子。在此，我们旨在确定 p75NTR 是否通过 BMSCs 介导骨愈合。

方法

通过流式细胞术（FCM）鉴定大鼠 BMSCs，以检测细胞周期和表面标志物。然后在 BMSCs 中转染 si/oe-p75NTR，随后进行茜素红染色以检测细胞的成骨分化，进行免疫荧光双重染色以检测 p75NTR 和分选素的表达，进行共免疫沉淀（CO-IP）以分析 p75NTR 和分选素之间的相互作用，以及 EdU 染色和细胞划痕实验以评估人脐静脉内皮细胞（HUVECs）的增殖和迁移。还检测了 HIF-1α、VEGF 和凋亡相关蛋白的表达。此外，构建了大鼠骨折愈合模型，并注射 BMSCs-si-p75NTR，然后使用 micro-CT 成像观察骨折情况，并评估血小板/内皮细胞黏附分子-1（CD31）的表达。

结果

结果表明成功分离出 BMSCs，p75NTR 抑制 BMSCs 的凋亡和成骨分化，而 si-p75NTR 导致 BMSCs 中分选素表达减少，HUVECs 的增殖和迁移增加，HIF-1α 和 VEGF 表达上调。此外，还观察到 p75NTR 和分选素之间存在相互作用。p75NTR 的敲低可减轻大鼠骨折的严重程度并增加 CD31 和成骨相关蛋白的表达。