人脐带间充质干细胞外泌体对大鼠骨折愈合的影响及其作用机制研究

Effect and mechanism of human umbilical cord mesenchymal stem cell derived exosomes on fracture healing in rats

摘要:

目的 观察人脐带间充质干细胞外泌体（hucMSC-Exos）对大鼠骨折愈合的影响，并探讨可能的作用机制。方法 取40只大鼠，成功复制股骨干骨折大鼠模型30只，随机分为对照组（注射PBS缓冲液50 μL、水凝胶100 μL至骨髓腔）、外泌体组（注射hucMSC-Exos 100μg、水凝胶100 μL至骨髓腔）、复合组（注射hucMSC-Exos 100 μg、水凝胶100 μL至骨髓腔，腹腔注射XAV939 1 mg/只），每组10只。干预后2周、4周行Micro-computed tomography活体扫描、生物力学测试最大载荷，Western blotting检测骨痂组织β-连环蛋白（β-catenin）、p-β-catenin、糖原合成酶激酶3β（GSK-3β）、Runx2蛋白的表达。结果 对照组、外泌体组、复合组大鼠干预后2周、4周骨小梁数量（Tb.N）、骨体积分数（BV/TV）比较，采用重复测量设计的方差分析，结果 ①不同时间点Tb.N、BV/TV有差异（F =23.584和31.267，均P =0.000）；②3组大鼠Tb.N、BV/TV有差异（F =85.512和54.796，均P =0.000），与对照组、复合组比较，外泌体组Tb.N较多，BV/TV较高，促进骨折愈合效果相对较好；③3组大鼠Tb.N、BV/TV变化趋势有差异（F =19.417和25.461，均P =0.000）。外泌体组最大载荷、最大载荷恢复率高于对照组和复合组（P <0.05）。与外泌体组比较，对照组组织学评分降低（P <0.05），复合组组织学评分升高（P <0.05）。与对照组比较，外泌体组p-β-catenin/β-catenin、Runx2蛋白相对表达量升高（P <0.05），GSK-3β蛋白相对表达量降低（P <0.05）；与外泌体组比较，复合组p-β-catenin/β-catenin、Runx2蛋白相对表达量降低（P <0.05），GSK-3β蛋白相对表达量升高（P <0.05）。结论 hucMSC-Exos可促进大鼠股骨干骨折愈合，其作用机制可能与激活Wnt/β-catenin信号通路有关。

Abstract:

Objective To observe the effect of human umbilical cord mesenchymal stem cell derived exosomes (hucMSC-Exos) on fracture healing in rats, and to explore the possible mechanism.Methods Thirty rats were established as the femoral shaft fracture models and were randomly divided into control group (bone marrow cavity injected with 50 μL of PBS and 100 μL of hydrogel), exosome group (bone marrow cavity injected with 100 μg of hucMSC-Exos and 100 μL of hydrogel), and combined group (bone marrow cavity injected with 100 μg of hucMSC-Exos and 100 μL of hydrogel, and intraperitoneally injected with 1 mg of XAV939), with 10 rats in each group. Micro-computed tomography scans were performed at 2 and 4 weeks after intervention. The maximum load of bones was measured via biomechanical approaches. The protein expressions of β-catenin, p-β-catenin, glycogen synthase kinase 3β (GSK-3β), and Runx2 in callus tissue were detected by Western blotting.Results Comparison of trabecular number (Tb.N) and bone volume fraction (BV/TV) among the control group, the exosome group, and the combined group at 2 and 4 weeks after intervention using repeated measures analysis of variance revealed that there were differences in Tb.N and BV/TV between different time points (F = 23.584 and 31.267, both P = 0.000) and among the groups (F = 85.512, 54.796, both P = 0.000). Compared with the control group and the combined group, Tb.N and BV/TV of the exosome group were higher, indicating a better effect of promoting fracture healing. The change trends of Tb.N and BV/TV were also different among the groups (F = 19.417 and 25.461, both P = 0.000). The maximum load and the recovery rate of the maximum load were higher in the exosome group than those in the control group and the composite group (P < 0.05). Compared with the exosome group, the histological score was lower in the control group (P < 0.05), but was higher in the combined group (P < 0.05). In comparison to the control group, the protein expressions of p-β-catenin/β-catenin and Runx2 were higher yet the protein expression of GSK-3β was lower in the exosome group (P < 0.05). Compared with the exosome group, the protein expressions of p-β-catenin/β-catenin and Runx2 were lower yet the protein expression of GSK-3β was higher in the combined group (P < 0.05).Conclusions The hucMSC-Exos play a role in promoting the fracture healing in rat models of femoral shaft fracture, and its mechanism may be related to the activation of Wnt/β-catenin signaling pathway.