Jmjd3和Ezh2拮抗调节小鼠骨折愈合

目的:探讨Jmjd3和Ezh2在小鼠骨折愈合过程中的作用。方法:以软骨细胞条件性基因敲除8-10周龄小鼠为研究对象,按基因型随机分为6组,每组5只:其中实验组基因型为Jmjd3~(fl/fl)/Col2a1-Cre ~(ERT2),Ezh2~(fl/fl)/Col2a1-Cre ~(ERT2)或Jmjd~(3fl/fl)/Ezh2~(fl/fl)/Col2a1-Cre ~(ERT2);对照组基因型为Jmjd3~(fl/fl),Ezh2~(fl/fl)或Jmjd3~(fl/fl)/Ezh2~(fl/fl)。建立骨髓腔中插入固定针的稳定性胫骨骨折模型,于骨折术后3天、5天和7天腹腔注射Tamoxifen 3 mg/次/天。各组于术后3W处死,并于骨折部位取材行X线片及组织学检查。结果:通过连续的X线影像学及HE组织切片观察,骨折术后3周是判断小鼠骨折愈合情况的最佳时间点。X线片发现骨折术后3W时软骨细胞内Jmjd3被敲除小鼠的骨折线较对照组明显且骨化骨痂大小和密度均较低,HE切片显示骨化骨痂面积显著低于对照组,而软骨骨痂面积高于对照组;相反,X线片发现Ezh2被敲除小鼠的骨痂面积明显大于对照组,且密度高于对照组,HE组织切片显示Ezh2被敲除的小鼠的骨化骨痂的钙化程度更高,骨小梁更粗更密集。最后,X线片和HE切片均没有发现软骨细胞Jmjd3和Ezh2同时被敲除的小鼠与对照小鼠之间存在明显差异。结论:以软骨细胞特异基因敲除小鼠为基础,我们首次发现Jmjd3具有促进骨折愈合的作用,而Ezh2具有抑制骨折愈合的作用;并且发现Jmjd3和Ezh2对抗调节小鼠的骨折愈合过程,这些发现为骨折愈合治疗提供了新的分子实验基础。

Jmjd3 and Ezh2 Antagonistically Regulate Fracture Healing of Bone in Mice

ABSTRACT Objective: To investigate the roles of Jmjd3 and Ezh2 in the processes of bone fracture healing in mice. Methods: Mice with gene conditional knockout (cKO)in chondrocytes were used in this study. Male mice at 8-10 weeks old were randomly divided into 6 groups according to their genotypes, with 5 mice in each group. Mice with Jmjd3 ^fl/fl /Col2a1-Cre ^ERT2, Ezh2 ^fl/fl /Col2a1-Cre ^ERT2 or Jmjd3 ^fl/fl /Ezh2 ^fl/fl /Col2a1-Cre ^ERT2 genotypes were experimental groups. Mice with Jmjd3 ^fl/fl , Ezh2 ^fl/fl or Jmjd3 ^fl/fl /Ezh2 ^fl/fl genotypes were con- trol groups. The mice were given a unilateral open tibiae transverse fracture operation with intramedullary needle fixation under aseptic condition. At 3, 5 and 7 days after the operation, the mice were intraperitoneally injected tamoxifen (3 mg per day for each time) and were sacrificed 3 weeks later for X ray and histology investigations. Results: X ray scanning and histology measurement showed that the most suitable time for observing the results of bone fracture healing in mice was at the third week after operation. At this stage, X ray showed that the fracture line in Jmjd3 cKO mice is clearer than the controlled group. Meanwhile, the ossified callus of Jmjd3 cKO group is much smaller and has lower density compared with the controlled group. HE tissue slices showed that the cKO group had significantly less ossified callus and more cartilage callus than the controlled group. In contrast, the X ray showed that the callus of Ezh2 cKO group was larger and had higher density than the controlled group. HE tissue slices showed that the calcification of ossified callus was higher in the cKO group. Besides, the trabecular bone of the cKO group was thicker and more dense. Lastly, the X ray and HE tissue slices showed that no significant difference were observed between Jmjd3/Ezh2 double cKO and controlled group. Conclusion: Based on gene cKO mice, we firstly detected that Jmjd3 can facilitate but Ezh2 suppress bone fracture healing in mice. We also find that Jmjd3 and Ezh2 counteractally regulate the process of bone fracture healing in vivo. These findings provide a new molecular experimental basis and im- plications for fracture healing treatment.