| Objective: Semaphorin3 A is the first Semaphorin identified in the vertebrate and acts to induce the retraction and collapse of the structure on axonal growth cone.Semaphorin3 A is a prototype of axonal guidance molecule in Semaphorin family,and recent studies have demonstrated the implication of Sema3 A in skeletal system.Neuropilin-1 are highly conserved single pass transmembrane proteins specifc to vertebrates.It was originally identifed as adhesion molecules in the nervous system,but were subsequently rediscovered as the ligand binding subunit of semaphorin3 A receptor in neurons and then as blood vessel receptors for the vascular endothelial growth factor VEGF.To investigate the mechanism of Sema3A/Nrp-1 in fracture healing after nerve injury by observing the expression of Sema3A/Nrp-1 in the tibia fracture healing after traumatic brain injury(TBI).Methods:A total of 192 Wistar female rats,8-10 weeks old and weighing 220-250 g,were randomly divided into tibia fracture group(group F),TBI group(group TBI),TBI with tibia fracture group(group TBI+F),and control group(group C).The tibia fracture model was established at the right side of group F;TBI model was made in group TBI by the improved Feeney method;the TBI and tibia fracture model was made in group TBI+F;no treatment was given in group C.The tissue samples were respectively collected at 3,5,7,14,21,and 28 days after operation;HE staining,immunohistochemistry staining,and Western blot method were used for the location and quantitative detection of Sema3 A in callus tissue.Results:ELISA results showed that the Sema3 A had the same expression trend during fracture healing in group F,group TBI and group TBI + F,and reached the highest level on the 3 day after injury.The group F reached the highest level in 14 days,but the group TBI and group TBI + F reached the highest level in 21 days,the peak was lower than that of the 3 day(P <0.05),followed by a downward trend,but still higher than the control group.HE staining showed that no obvious changes were observed at each time point in group TBI and C.At 3 and 5 days,there was no obvious callus growth at fracture site with inflammatory cells and fibrous tissue filling in groups Fand TBI + F.At 7 and 14 days,fibrous tissue grew from periosteum to fracture site in groups F and TBI + F;the proliferation of chondrocytes in exterior periosteum gradually formed osteoid callus at fracture site in groups F and TBI + F.The chondrocyte had bigger size,looser arrangement,and more osteoid in group TBI + F than group F.Group TBI had disorder periosteum,slight subperiosteal bone hyperplasia,and no obvious change of bone trabecula in group B when compared with group C.At 21 and 28 days,cartilage callus were gradually replaced by new bone trabecula in groups F and TBI + F.Group TBI + F had loose arrange,disorder structure,and low density of bone trabecula,big callus area and few chondrocyte and osteoid in group TBI + F when compared with group F;group TBI was similar to Group C.Immunohistochemistry staining showed that Sema3 A expression in chondrocytes in group TBI + F was higher than that in group F,particularly at 7,14,and 21 day.Sema3 A was significantly higher in osteoblasts of new bone trabecula in group F than that in group TBI + F,especially at 14 and 21 days(P<0.05).Western blot results showed that the Sema3 A had the same expression trend during fracture healing in groups F and TBI + F.However,the expression of Sema3 A protein was significantly higher in group TBI + F than that in group F(P<0.05)and in group TBI than group C(P<0.05)at 7,14,21,and 28 days.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. |
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