【摘要】 目的：分离得到中华真地鳖的活性组分并探讨其对骨折愈合的影响及其作用机制。方法：采用改良的Einhorn法建立大鼠胫骨骨折模型。使用放射学方法评估骨折愈合的进展情况。翻红固绿染色和CD31免疫组化染色来评估骨折部位骨细胞的生长和血管生成情况。MTT方法和细胞伤口愈合方法分别用于分析细胞的活力和迁移能力。采用Transwell法检测细胞的侵袭能力，小管形成实验用于评估人血管的血管生成能力。q RT-PCR用于评估基因转录水平的变化。结果：土鳖虫组分3(TF3)显著缩短模型大鼠的骨折愈合时间。X射线结果显示，在第14天，TF3治疗组的骨折愈合情况明显优于对照组（P=0.0086）。组织染色结果显示TF3治疗组骨折部位的软骨生长情况和H型血的数量优于对照组。在体外，TF3显著促进MC3T3-E1和HUVECs细胞增殖和伤口愈合（均P <0.01）。Transwell分析显示TF3促进HUVECs的迁移但抑制MC3T3-E1细胞的迁移。小管形成实验证实TF3显著增加了血管内皮细胞形成微管的能力。q RT-PCR分析显示TF3显著促进了HUVECs中VEGFA、SPOCD1、NGF和NGFR的转录。在MC3T3-E1细胞中，TF3处理显著升高了RUNX2和COL2A1的转录水平。结论：TF3通过促进与RUNX2通路相关的骨再生以及与VEGFA通路相关的血管生成从而加速骨折愈合。更多还原

【Abstract】 Objective: To determine the active components of Eupolyphaga sinensis Walker(Tu Bie Chong) and explore the mechanisms underlying its fracture-healing ability.Methods: A modified Einhorn method was used to develop a rat tibial fracture model. Progression of bone healing was assessed using radiological methods. Safranin O/fast green and CD31 immunohistochemical staining were performed to evaluate the growth of bone cells and angiogenesis at the fracture site. Methylthiazoletetrazolium blue and wound healing assays were used to analyze cell viability and migration. The Transwell assay was used to explore the invasion capacity of the cells. Tubule formation assays were used to assess the angiogenesis capacity of human vascular endothelial cells(HUVECs). q RTPCR was used to evaluate the changes in gene transcription levels.Results: Tu Bie Chong fraction 3(TF3) significantly shortened the fracture healing time in model rats. X-ray results showed that on day 14, fracture healing in the TF3 treatment group was significantly better than that in the control group(P =.0086). Tissue staining showed that cartilage growth and the number of H-shaped blood vessels at the fracture site of the TF3 treatment group were better than those of the control group.In vitro, TF3 significantly promoted the proliferation and wound healing of MC3T3-E1s and HUVECs(all P <.01). Transwell assays showed that TF3 promoted the migration of HUVECs, but inhibited the migration of MC3T3-E1 cells. Tubule formation experiments confirmed that TF3 markedly promoted the ability of vascular endothelial cells to form microtubules. Gene expression analysis revealed that TF3 significantly promoted the expression of VEGFA, SPOCD1, NGF, and NGFR in HUVECs. In MC3T3-E1 cells, the transcript levels of RUNX2 and COL2A1 were significantly elevated following TF3 treatment.Conclusion: TF3 promotes fracture healing by promoting bone regeneration associated with the RUNX2pathway and angiogenesis associated with the VEGFA pathway.更多还原