Effect Of Antler Stem Cell Secretion On Promoting Osteogenesis And Inhibiting Osteoclast

Osteoporosis is the most common metabolic bone disease.As people age,the incidence of osteoporosis gradually increases,so does the incidence of fractures,which can cause serious harm to the health of patients.Osteoporosis is caused by a variety of factors.Its basic pathological mechanism is the clinical symptom caused by the defects of the coupling between bone resorption and bone formation in the process of bone metabolism,which leads to the imbalance of calcium and phosphorus metabolism in human body and the gradual reduction of bone density.Osteoporosis is more common in postmenopausal women.Currently,existing drugs can only prevent and delay OP,and cannot reach the root cure effect and other shortcomings.Therefore,the research and development of new drugs for osteoporosis has become the research focus of scholars at home and abroad.The antler is the unossified,feathery young horn of a male deer(Cervus nippon)or red deer(Cervus canadensis)on its head.According to traditional Chinese medicine,Antler velvet has the function of tonifying kidney and strengthening Yang,producing lean blood,replenishing marrow and strengthening bones.These findings are not only the result of the traditional application of traditional Chinese medicine,but also highly related to the biological characteristics of antler: Antler is the only bone organ in mammals that can completely regenerate after loss.Deer antler has an overspeed growth period every year.Studies have shown that the overspeed growth of deer antler is caused by the cells and the microenvironment in which it resides.In particular,substances secreted by the stem cells of velvet antler are the basis for the overspeed growth of bone tissue.However,whether these secretions can be effective on bone cells of other animals,including humans,has not been reported.Therefore,in this paper,the mechanism of action of Antler stem cell conditioned medium(ASC-CM)on osteoporosis was described from the above two aspects based on cell level studies and animal level studies,so as to provide scientific basis for further development and utilization of Antler for medicinal use.In this study,the medium of Antler RM cells was collected and filtered through0.22μm filter membrane to obtain pure ASC-CM.The osteoporosis model of adult SD female rats was established by ovariectomy in vitro.Bone marrow mesenchymal stem cells(BMSCs)and bone marrow mononuclear cells(BMMs)were obtained from the shin bones of adult SD rats,which were washed out of the bone marrow with PBS buffer solution under sterile conditions and collected.BMSC was cultured with ASCCM,and the optimal concentration was determined to be 0.2μg/μl by CCK-8 method,which was used for subsequent experiments.1.Cultured rat BMSC with ASC-CM obtained above.The proliferation effect of ASC-CM on rat BMSC was analyzed by EDU,CCK-8 and cell scratch assay.The results showed that 0.2μg/μl ASC-CM had obvious proliferation effect on BMSCs in rats,which was higher than that in positive control group.It provides a reference for the follow-up experimental research.2.The rat BMSC was cultured with ASC-CM and induced by osteogenic induction solution.The relative m RNA expression levels of BSP,COL-I,OCN,OPN and RUNX-2 were detected by alizarin red staining,alkaline phosphatase(ALP)staining and q PCR.The relative expressions of TRAP,OC,BALP and other osteoblast proteins were analyzed by Western Blot and WB.It was concluded that ASC-CM significantly promoted the osteogenic differentiation of BMSC.This theory provides a theoretical basis for the verification of subsequent in vivo experiments.3.The BMMs obtained above were co-cultured with ASC-CM,25ng/ml macrophage colony-stimulating factor(M-CSF)and 30ng/ml nuclear factor κB receptor activating factor ligand(RANKL).Through observation,it can be concluded that ASC-CM can inhibit the aggregation of monocytes into osteoclasts,further verifying that ASC-CM can alleviate the occurrence of OP.4.The above in vitro studies have shown that ASC-CM can promote osteogenic differentiation of BMSC and inhibit osteoclastic differentiation of BMMs.To provide the theoretical basis for animal experiments of this step.The relative content of serum alkaline phosphatase in rats was determined by enzyme linked immunosorbent assay(ELISA),and the expression of RUNX-2 and Os X relative proteins in rats was verified by WB test.The results showed that ASC-CM had an obvious effect on OP rats,and the mechanism might be related to Wnt/β-catenin signaling pathway.Results: The cell experiments showed that the secretion of antler stem cells could not only promote the proliferation and osteogenic differentiation of rat BMSCs,but also inhibit the osteoclastic differentiation of rat BMMs.The experiments on OP model rats showed that the secretion from velvet antler stem cells could effectively improve OP,and the osteogenic factors such as OC and BALP were increased,while the content of osteoclast factor TRAP was significantly decreased.The expression of osteoblastrelated proteins was also significantly increased.