| Osteoporosis is a common and complex bone metabolic disease disease that is characterized by a reduction in bone mineral density (BMD) and a microarchitecture deterioration of bone tissue with a consequent increase of fracture risk. The most common fractures are morphometric vertebral fracture, distal radius fracture and others in clinical practice and may cause kyphosis, shorten body and respiratory diseases.Patients often have to complete bed rest for a long time, the complication such as bedsore, pulmonary infection, urinary tract infection diseases can raise the risk of die. Therefore we need to further understand the pathogenesis of osteoporosis and osteoporotic fracture healing mechanism.(l)This study aimed to investigate the association of the g.27563G>A osteoprotegerin (OPG) genetic polymorphism with bone mineral density (BMD) and osteoporosis. In order to further understand the pathogenesis of osteoporosis, provide support for the treatment of osteoporosis and its complications.A case-control study was carried out with 435 osteoporosis postmenopausal women cases and 442 age-matched healthy controls. The BMD at the femoral neck hip, lumbar spine (L2-4), and total hip were assessed by Norland XR-46 dual-energy X-ray absorptiometry. The genotypes of the g.27563G>A genetic polymorphism were detected by created restriction site-polymerase chain reaction and verified by DNA sequencing methods. We detected that the g.27563G>A genetic polymorphism was a non-synonymous mutation that resulted in an arginine (Arg) to glutamine (Gin) amino acid replacement (p.Arg333Gln). Significant differences were found in the BMD of the femoral neck hip, lumbar spine (L2-4), and total hip among different genotypes of the g.27563G>A genetic polymorphism. Subjects with the genotype GG had significantly higher BMD values than those with genotypes GA and AA (P< 0.05). Our data indicated that the A allele of the g.27563G>A genetic polymorphism in OPG could be associated with lower BMD values in the Chinese postmenopausal women evaluated, and that it might be an increased risk factor for osteoporosis.(2) Investigate the influence of OPG gene polymorphism on protein expression and biological activity. In the present study, Our data indicated that the a allele of the g.27563G>A genetic polymorphism in OPG could be associated with lower BMD values in the Chinese postmenopausal women evaluated, and that it might be an increased risk factor for osteoporosis. However, the relationship between OPG gene polymorphism and osteoporosis is not clear. This part,we research the influence of OPG gene mutation on protein expression and activity, on the basis of the successful build a cell culture system of expression of wild type and mutant of OPG gene In vitro.Results showed that the genetic mutations of OPG can not influence the protein expression level of OPG, but can inhibit the normal activity of the protein.(3) Investigate the signal pathway involved in the effect of OPG gene mutation on osteoclast differentiation. M-CSF and RANKL were used to induce RAW264.7 cells, and the wide type OPG, mutant type OPG, SB202190, SP600125,2-APB or KN93 were added to the cells. The phosphorylation levels of p38, JNK, ERK and CaMK ? were detected by western blotting. The intracellular level of Ca2+ was detected by flow cytometry. The results showed that wide type OPG and mutant type OPG inhibited the phosphorylation levels of p38, JNK and ERK. However, the inhibition effect of wide type OPG on p38 and JNK was higher than mutant type OPG, the inhibition effect of wide type OPG on ERK was similar to mutant type OPG. Wide type OPG and mutant type OPG inhibited the phosphorylation levels of CaMK ? and intracellular level of Ca2+. There was no significant difference between the effect of wide type OPG and mutant type OPG. Our data indicated that the p38 and JNK signal pathways were involved in the effect of OPG gene mutation on osteoclast differentiation.(4) Osteoporosis is characterized by a high risk of fracture.The clinical expression of osteoporosis is a fragility fracture. The most common fractures are morphometric vertebral fractures and distal radius fractures, among others.A case-control study was carried out with 102 postmenopausal women with osteoporosis fractures,102 postmenopausal women with osteoporosis and 102 healthy women of similar age. After fasting,5 ml of venous blood was extracted from each of the subjects in order to observe changes to the nerve growth factor caused by the ELISA method. It was found that the NGF level in the serum declined in the postmenopausal osteoporosis and osteoporosis fracture women (P< 0.05), which indicates that a decrease in the NGF level of the serum may be a cause of postmenopausal osteoporosis and osteoporosis fractures in women.(5) The regulation of the expression of bone morphogenetic protein (BMP) of nerve growth factor (NGF) was investigated with analysis of the mechanism of action of nerve growth factor in promoting fracture repair. Methods:108 healthy New Zealand white rabbits were randomly divided into three groups:the NGF application group (Group A), the antagonistic NGF group (Group B) and the control group (Group C). Each group contained 36 rabbits. Standard models of right mid-piece radial fractures of rabbits were made. Rabbits of group A accepted a intravenous injection of NGF 200ng/Kg twice a day, and the same dose was administered for 1 week continuously; group B received intraperitoneal injections of NGF antibody 400ng a time, three times a day for three weeks; group C was injected with physiological saline 1ml at a time, twice a day for three weeks.6 rabbits in each group were sacrificed at 24h,48h, 1week,3weeks,6weeks and 8 weeks, respectively. The BMP proteins of the fracture specimens were analyzed by Western immunoblotting. Results:1. Applying NGF promoted the expression of BMP in fracture, while applying an NGF antagonist inhibited the expression of BMP.2. The group that was given NGF produced more BMP earlier than the control group or the NGF antagonist group during fracture healing, while the control group produced more BMP earlier than the NGF antagonist group (P<0.05). Conclusion:The results indicate that NGF can promote the expression of BMP in radial fracture of rabbits, which can be directly or indirectly beneficial to fracture healing.(6) The effect of nerve growth factor on vascular endothelial growth factor expression during the process of fracture healing in rabbits was investigated. A radial fracture model was established in white rabbits, which were then divided into three groups, which were treated with nerve growth factor, nerve growth factor monoclonal antibody, and saline respectively. These groups served as the nerve growth factor group, antagonizing nerve growth factor group, and control group. The vascular endothelial growth factor expression was detected with western blot analysis at 24 hours,48 hours,1 week,3 weeks,6 weeks and 8 weeks post-injury. The results showed that the expression level was the highest in the nerve growth factor group, then in the control group, and it was the lowest in the antagonizing nerve growth factor group (P< 0.05). The experimental findings suggest that nerve growth factor can promote the expression of vascular endothelial growth factor in the fracture healing process.In summary, Our data indicated that the A allele of the g.27563G>A genetic polymorphism in OPG could be associated with lower BMD values in the Chinese postmenopausal women evaluated, and that it might be an increased risk factor for osteoporosis. Further study to investigate the effect of OPG gene mutation on protein expression and its biological activity. The results showed that the mRNA and protein levels of OPG between wide type OPG and mutant type OPG were smilar. Wide type OPG and mutant type OPG inhibited osteoclast differentiation and bone resorption activity, however, the inhibition effect of wide type OPG was higher than mutant type OPG. Our data indicated that OPG gene mutation could affect osteoclast differentiation and bone resorption activity. Our data indicated that the p38 and JNK signal pathways were involved in the effect of OPG gene mutation on osteoclast differentiation. Nervous system is closely related to the bone tissue and regulate the normal metabolism of bone and bone fracture repair by the neurotransmitters, peripheral nerve synapses and hormones. In the last three parts of the study, Our data indicated that Serum levels of nerve growth factor associated significantly with the osteoporosis and osteoporotic fracture.The mechanism of nerve growth factor promoting fracture healing is to promote the expression of bone morphogenetic protein and vascular endothelial growth factor during the process of fracture healing. This indicate that Nerve growth factor play an important role in the preventing and treating of osteoporosis and osteoporotic fracture. |
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