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目的通过观察脑信号蛋白3A(Semaphorin 3A,Sema3A)在创伤性颅脑损伤(traumatic brain injury,TBI)后胫骨骨折愈合中的表达变化,探讨Sema3A在神经损伤后骨折愈合中的作用机制。方法取8~10周龄Wistar雌性大鼠192只,体质量220~250 g,随机分为胫骨骨折组(A组)、TBI组(B组)、TBI伴胫骨骨折组(C组)、对照组(D组),每组48只。A组制备右侧胫骨骨折模型,B组采用改进后的Feeney法自由落体颅脑损伤装置制备TBI模型,C组制备TBI合并胫骨骨折模型,D组不作处理。分别于术后3、5、7、14、21、28 d处死8只动物取材,采用HE染色、免疫组织化学染色及Western blot法对骨痂组织中Sema3A进行定位、定量检测。结果 HE染色示D组各时间点未见明显变化。术后3、5 d,A、C组骨折断端无明显骨痂生长,可见炎性细胞及纤维组织填充;B组与D组无差异。7、14 d,A、C组纤维组织自骨膜下向断端间隙生长,骨外膜软骨细胞增生,逐渐向断端生成软骨痂,C组与A组相比骨外膜软骨细胞体积大、排列疏松,类骨质多;B组与D组相比,骨膜稍紊乱,骨膜下骨质轻度增生,骨小梁未见明显差异。21、28 d,A、C组软骨痂逐渐被新生骨小梁代替,C组与A组相比,骨小梁排列疏松、结构紊乱、密度低,骨痂面积较大,残存少量软骨细胞及类骨质;B组与D组比较无明显差异。免疫组织化学检测示,Sema3A阳性染色在C组软骨细胞表达高于A组,尤其在7、14、21 d;Sema3A阳性染色在A组新生骨小梁成骨细胞表达较C组高,尤其在14、21 d;差异均有统计学意义(P<0.05)。Western blot检测示,Sema3A在A、C组骨折愈合过程中表达趋势相同,术后7、14、21、28 d C组Sema3A蛋白相对表达量高于A组(P<0.05);B组Sema3A蛋白相对表达量在术后7、14、21、28 d显著高于D组(P<0.05)。结论 Sema3A在神经损伤后骨折愈合中表达异常,可能通过促进软骨细胞增殖、降低感觉神经纤维分布及成骨细胞分化,在神经损伤后骨折愈合中发挥作用。
Objective To investigate the effect of Sema3A on fracture healing after traumatic brain injury (TBI) by observing the expression of Sema3A in fracture healing after traumatic brain injury (TBI). Methods Ninety-two Wistar female rats aged 8-10 weeks were divided into three groups randomly: tibial fracture group (group A), TBI group (group B), TBI group with tibia fracture (group C), and control group Group (Group D), 48 rats in each group. The right tibial fracture model was prepared in group A, the TBI model was prepared by modified Feeney free-fall craniocerebral injury device in group B, and the tibial fracture model was prepared in group C without treatment. Eight animals were killed at 3, 5, 7, 14, 21 and 28 days after operation respectively. HE staining, immunohistochemistry and Western blot were used to locate and quantitatively detect Sema3A in callus. Results HE staining showed no significant changes in D group at each time point. At 3 and 5 days after operation, there was no obvious callus growth in the fracture sites of A and C groups, and the infiltration of inflammatory cells and fibrous tissue was observed. There was no difference between group B and group D. At 7 and 14 days, the fibrous tissues of group A and C grew from the subperiosteal to the interphalangeal, the cartilage cells proliferated and gradually formed the cartilage callus. The extracellular chondrocytes in group C were larger than those in group A, Loosely arranged, osteoid and more; Group B compared with the D group, the periosteum slightly disturbed, subperiosteal bone mild hyperplasia, trabecular bone no significant difference. At 21 and 28 d, the cartilage scab in group A and group C was gradually replaced by newborn trabecular. Compared with group A, group C had loose trabecular arrangement, disorganized structure, low density, large callus area, few remaining chondrocytes, Bone-like; B group and D group no significant difference. Immunohistochemistry showed that the expression of Sema3A in group C was higher than group A, especially at 7, 14 and 21 days. The expression of Sema3A in group A was higher than that in group C, especially in group A 14 and 21 days respectively. The differences were statistically significant (P <0.05). Western blot showed that the expression of Sema3A was similar in group A and group C, and the relative expression of Sema3A protein in group C was higher than that in group A at 7, 14, 21 and 28 days after operation (P <0.05). Sema3A protein The relative expression levels at 7, 14, 21 and 28 d after operation were significantly higher than those in group D (P <0.05). Conclusion The abnormal expression of Sema3A in fracture healing after nerve injury may play a role in fracture healing after nerve injury by promoting the proliferation of chondrocytes, decreasing the distribution of sensory nerve fibers and the differentiation of osteoblasts.