| Objective:Based on the regulation of Slit-Robo signaling pathway on CD31hiEmcnhi coupled osteogenesis,the mechanism of Chinese medicine tonifying kidney on induced membrane technique bone reconstruction was analyzed.Methods:It is expected to purchase 12 male SD rats（SPF grade）for the preliminary experiment,weighing about 180-200 g.The rats are kept in cages with 4 rats per cage.The air conditioning temperature of the animal house is set at 26℃.After one week of adaptive feeding,the body weight increased to about 250 g.After the pre-experiment,48male SD rats were purchased again,and the arrangement of rats was the same as before.Excel software was used for body weight stratification randomized block design,and all rats were randomly divided into 4groups in order of body weight:blank group（group A）,model group（group B）,Chinese medicine medium dose group（group C）,Chinese medicine high dose group（group D）,with 12 rats in each group（previous experiments have shown that Chinese medicine medium and high dose groups have exact effects on inducing membrane）.The induced membrane model was established in one stage,the skin was cut,the femur was exposed,and the bone was accurately excised.Except for the blank group,the other three groups were placed with bone cement,washed and sutured,and antibiotics were injected for five consecutive days after operation,6 weeks after the bone cement was removed,the bone graft was opened.In the second stage of modeling,two tailbones were cut into pieces according to the steps,and then the femur plate was exposed,the bone cement was removed,the fractured tailbone was implanted,and the wound was sutured.On the second day after the bone cement was removed,the Chinese medicine groups were given different concentrations of total flavonoids of rhizoma drynariae,with the medium concentration of 78.75·kg mg-1·d-1 and the high concentration of 157.5·mg mg-1·d-1.The rats in the blank group and model group were given the same amount of pure water by intragastric administration（2ml per 500g body weight as expected）.Imaging examination was performed8 weeks after surgery,and the expressions of CD31,Emcn,slit3 and ROBO related genes were detected by RT-PCR.Results:All the rats did not die,their life behavior did not change abnormally,and their body weight increased gently over time.The skin sutures of two rats were torn open one day after surgery,resulting in partial exposure of muscles.The wounds healed well after re-disinfection;in rats with wound infection,the number of days of antibiotic injection was increased and the wound could heal quickly,and there were no complications such as plate sliding,screw loosening and removal,or femoral malformation and movement disorders in rats.X-ray results showed a small amount of callus formation in the blank group,no loosening of plate screws,and the recovery degree of bone defect area was about 25%.The bone composition of the model accounted for about 50%of the bone defect area,the plate screw was firm without falling off,part of the fracture line disappeared,the cortical bone was rebuilt but weak.In the Chinese medicine medium dose group,the osteotomy area was close to more than 75%,the screws and plates were intact,the fracture line was blurred,and a little cortex inside the bone was not continuous.In the high-dose Chinese medicine group,the bone formed full of defect area,the femur end was straight,the plate was not distorted,the bone was good and no erosion image,the fracture line disappeared completely,and cortical bone reconstruction was more than 95%.PCR showed that m RNA expressions of CD31,Emcn,slit3 and ROBO in the model group were higher than those in the blank group,and the difference was statistically significant（P<0.05）;However,the expression of related genes in Chinese medicine medium and high dose groups was significantly up-regulated in comparison model group,with statistical difference（P<0.05）,the gene expression of the high-dose group was also partially increased compared with the medium-dose group.Conclusion:Total flavonoids of rhizoma drynariae regulate vascular coupling osteogenesis through slit-Robo signaling pathway to promote induced membrane bone reconstruction.This study confirms the correlation between CD31hi Emcnhi vaso-coupled osteogenesis and Slit-Robo signaling pathway during induced membrane bone tissue reconstruction,and initially reveals the molecular biological mechanism of total flavonoids of rhizoma drynariae in promoting secondary bone reconstruction induced by membrane technology. |
