神经毡蛋白-1在创伤性颅脑损伤伴胫骨骨折愈合过程中的表达变化

Nrp-1 expression in healing process of traumatic brain injury combined with tibial fracture

目的:观察创伤性颅脑损伤(traumatic brain injury,TBI)后神经毡蛋白-1(neuropilin-1,Nrp-1)及其受体脑信号蛋白3A(semaphorin 3A,Sema3A)、VEGF在胫骨骨折愈合中的表达变化,探讨Nrp-1在神经损伤后病理性骨痂形成中的作用机制。方法:雌性Wistar大鼠192只,8~10周龄,体重220~250 g,随机分为对照组(C组)、胫骨骨折组(F组)、TBI组、TBI合并胫骨骨折组(TBI+F组),每组48只。每组术后3,5,7,14,21及28 d处死8只大鼠取材。对骨痂中的Nrp-1行免疫组织化学及Western印迹定位、定量检测,对Sema3A及VEGF行Western印迹定量检测。结果:免疫组织化学显示Nrp-1在TBI+F组骨外膜成骨区域及软骨内成骨区域的软骨细胞中的表达较F组增高,尤其在术后7,14和21d(均P<0.05);Nrp-1在F组新生骨小梁周围成骨细胞中的表达较TBI+F组高,尤其在术后14和21 d(均P<0.05);Western印迹检测表明:Nrp-1,Sema3A和VEGF在TBI+F组、F组骨折愈合过程中表达趋势相同,但在各时间点TBI+F组Nrp-1表达量高于F组,下降缓慢,尤其在术后14,21和28 d(均P<0.05),同时TBI+F组Sema3A/VEGF比率高于F组,差异有统计学意义(P<0.05)。结论:神经损伤后Nrp-1在骨折愈合过程中呈异常表达,可能通过促进软骨细胞分化与增殖、阻止神经纤维在软骨痂的长入、降低成骨细胞分化,参与神经损伤后病理性骨痂的形成。

Objective： To examine the expression of neuropilin-1 （Nrp-1）, semaphorin 3A （Sema3A） and vascular endothelial growth factor （VEGF） in the healing process of tibial fracture after traumatic brain injury and to explore the mechanism of Nrp-1 in the formation of pathological callus after nerve injury. Methods： A total of 192 Wister female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into a control group （Group C）, a tibia fracture group （Group F）, a traumaticbrain injury group （Group TBI）, a traumatic brain injury combined with the tibia fracture group （Group TBI＋F） （n=48 in each group）. Tissue samples were collected at 3, 5, 7, 14, 21 and 28 days, respectively （n=8 for each time point）. The expression of Nrp-1 in callus tissues were examined by immunohistochemistry and Western blot, while the expression of Sema3A and VEGF were detected by Western blot. Results： Compared with the Group F, the expression of Nrp-1 in chondrocytes of bone formation area and cartilage area was higher in the Group TBI＋F, particularly at the 7th , 14th and 21st day （P〈0.05）, while the expression of Nrp-1 in osteoblast cells of fresh bone trabecula region was lower in the Group TBI＋F, particularly at the 14th and 21st day （both P〈0.05）. Western blot showed that the expression ofNrp-1, Sema 3A and VEGF had the same trends in the Group TBI＋F and the Group F. However, at each time point, the expression of Nrp-1 in the Group TBI＋F was significantly higher and slowly decreased, particularly at the 14th, 21st and 285 day （all P〈0.05）. Meanwhile, the ratio of Sema 3A/VEGF in the Group TBI＋F was significantly higher than that in the Group F, with statistical difference （P〈0.05）. Conclusion： The Nrp-1 is expressed abnormally in the process of fracture healing after nerve injury.It may play a role in the formation of pathological callus after nerve injury by promoting the preliminary and proliferation of chondrocytes, and inhibiting the growth of nerve fibers in the soft callus as well as the differentiation of osteoblast cell.