Study On The Role Of Slit2 In Promoting Osteogenic Differentiation

BackgroundOsteoporosis is a bone disease characterized by decreased bone mineral density and deterioration of bone structure,which seriously threatens human health.Osteoporosis includes primary osteoporosis and secondary osteoporosis.Primary osteoporosis includes osteoporosis in senile and postmenopausal women,and secondary osteoporosis caused by chronic inflammatory diseases,including rheumatoid arthritis,ankylosing spondylitis,inflammatory bowel disease,etc.The treatments for osteoporosis include estrogen therapy,calcitonin therapy and zoledronic acid therapy.Zoledronic acid is currently widely used,but it causes serious side effects such as femoral fracture and jaw osteonecrosis.Therefore,it is an urgent problem to find a new target for the treatment of osteoporosis.Recent studies have found that factors related to the nervous system such as SEMA promote the formation of bones,and the vascular regulation system also plays an important role in the skeletal system.Slit2 acts as an axon guide molecule in the nervous system and participates in the formation of blood vessels,but its specific role and mechanism in the skeletal system have not been reported.ObjectiveIn this study,Slit2-Tg mice are used to carry out general research on bone quality,and use primary bone marrow mesenchymal stem cells to induce osteogenic differentiation to explore the effect of Slit2 on osteogenic differentiation and its specific mechanism.Material and MethodsFirstly,Slit2-Tg mice were identified by PCR method.The mice were grouped according to their age and sex.WT female mice at 8 weeks,24 weeks,36 weeks,and48 weeks,male mice at 8 weeks,and 48 weeks were used as control groups.Slit-Tg female mice at the age of 8 weeks,24 weeks,36 weeks,and 48 weeks,and male mice at the age of 8 weeks and 48 weeks were the experimental groups,each with 4 mice,scanned by Micro-CT Analysis and HE staining method to observe the bone quality of the distal femoral metaphysis of 8-week-old,24-week-old,36-week-old and48-week-old mice,and detect the bone quality of the fourth lumbar vertebra of48-week-old female mice by Micro-CT Situation: Primary bone marrow mesenchymal stem cells were extracted from 8-weeks-old WT female mice and Slit2-Tg female mice,and osteogenic differentiation was induced and then stained with alkaline phosphatase and alizarin red for osteogenic ability The detection of bone marrow macrophages was extracted for osteoclast differentiation and then Trap staining was used to detect the osteoclast capacity.In order to further explore the ability of Slit2 to promote osteogenic differentiation,the bone marrow mesenchymal stem cells derived from two kinds of mice were osteoinduced for 7 days and then sequenced,so as to preliminarily explore the mechanism of Slit2 in the osteogenic differentiation of bone marrow mesenchymal stem cells;through XF the seahorse experiment detects the mitochondrial metabolism in the process of osteogenic differentiation.Results(1)Slit2-Tg female mice at the age of 8 weeks,24 weeks,36 weeks,and 48 weeks have better femoral bone quality than WT mice,and the vertebral bone quality of 48-week-old Slit2-Tg female mice is better than that of WT mice.(2)Slit2-Tg male mice at the age of 8 weeks and 48 weeks have better femoral bone quality than WT mice;(3)The difference in bone quality between the two mice is more obvious in female mice;(4)BMSCs derived from Slit2-Tg mice have better osteogenic ability,and BMSCs derived from Slit2-Tg mice inhibit the formation of osteoclasts;(5)Slit2 may participate in the process of osteogenic differentiation by regulating the metabolism of mitochondria.ConclusionGeneral experiments show that Slit2 can promote the formation of bone in the body,and can prevent senile osteoporosis;Slit2 can promote the osteogenic differentiation of bone marrow mesenchymal stem cells and the formation of mineralized nodules;Slit2 inhibits the formation of osteoclasts;Slit2 may participate in the osteogenic differentiation process of bone marrow mesenchymal stem cells by regulating the metabolic process of mitochondria.