The Effect And Mechanism Of S100A10 On The Proliferation And Differentiation Of Antler Precartilaginous Cells

Deer antler is the only special bone organ in mammals that can be periodically and completely regenerated in years.The way of bone formation is very similar to that of human beings,which is through endochondral ossification.However,compared with human beings,the growth rate of antler is extremely fast,far exceeding the proliferation rate of cancer cells,but no canceration occurs.These biological phenomena of velvet antler show its strong proliferation and differentiation function,making it an ideal model for studying bone regeneration in mammals.The rapid growth of antler and bone regeneration are achieved by continuous proliferation and differentiation and maintaining dynamic balance under the action of various cytokines.So far,people have not been able to completely solve the mystery of antler growth.S100 calcium binding protein A10(S100A10),a unique member of the S100 family,is widely involved in and regulates cell proliferation and differentiation.Studies have shown that S100A10 is a unique mineralization gene of antler cells and is highly expressed in antler tissues.However,the expression level of S100A10 in different tissue layers of antler tip is not clear,and the specific effect of this gene on the proliferation and osteogenic differentiation of antler cells and the osteogenic mechanism have not been reported.Therefore,this study further clarified the expression level of S100A10 in different tissue layers of antler tip,and revealed the effect of S100A10 on the proliferation and osteogenic differentiation of antler cells and the mechanism of S100A10 on the osteogenic differentiation of antler cells at the molecular level.The results will provide new ideas for clinical effective treatment of bone defects and wound repair.1.Real-time fluorescence quantitative polymerase chain reaction(q RT-PCR),Western blot and immunofluorescence staining were used to analyze the expression level of S100A10 in the apical growth center of antler : Reserve mesenchyme(RM)layer,Precartilage(PC)layer and Cartilage(CA)layer.The experimental results showed that the expression of S100A10 gene was the highest in the PC layer,followed by the CA layer and the lowest in the RM layer among the three tissue layers at the top of antler.2.The S100A10 interference vector was constructed and transfected into 293 T cells by 3 plasmid system for lentivirus packaging.Firstly,three high-resolution sh RNA oligonucleotide sequences were designed for the sika deer S100A10 sequence,and homologous recombination primers with carrier homologous arms,enzyme digestion sites,and sh RNA partial fragments were designed.The ligation product was transformed into E.coli competent cells,and identified by bacterial PCR and sequencing to complete the construction of interference vector.Then,the positive interference vector and the empty vector were packaged in 293 T cells using the envelope plasmid p MD2.G and the packaging plasmid p SPAX2.The results showed that the S100A10 lentiviral interference vector was successfully constructed and the lentiviral packaging of the S100A10 interference vector was completed.3.In order to explore the effect of S100A10 on the proliferation and osteogenic differentiation of antler PC cells,S100A10 gene in antler PC cells was silenced by RNA interference technology.Firstly,PC cells were isolated and cultured from antler PC tissues,and then PC cells were infected with successfully packaged S100A10 interference lentivirus.Positive cells were screened by puromycin to obtain stable cell lines.The interference efficiency of S100A10 was detected by q RT-PCR and Western blot,and the group with the highest interference efficiency was used for subsequent experiments.CCK8 assay was used to detect the effect of S100A10 interference on the proliferation of PC cells in vitro.The changes of PC cell cycle were detected by flow cytometry.Alkaline phosphatase(ALP)staining and ALP activity were used to detect the early osteogenic differentiation of cells.Alizarin red staining and quantitative analysis were used to detect the late osteogenic differentiation of cells.The m RNA expression levels of osteogenesis-related genes : osteocalcin(OCN),osteopontin(OPN)and type I collagen(COL-1)and BMP-2 signaling pathway-related genes : bone morphogenetic protein-2(BMP-2),Runt-related transcription factor 2(Runx2),signal transduction molecules Smad1 and Smad5 were detected by q RT-PCR.The expression levels of BMP-2,Runx2,Smad1 and Smad5 were further verified by Western blot.The results showed that after S100A10 interference,the G0/G1 phase of PC cells increased significantly(*P<0.05),the proportion of S phase decreased significantly(*P<0.05),and there was no significant difference in G2/M phase.CCK8 test also showed that S100A10 interference could reduce the proliferation ability of PC cells in vitro.In addition,the ALP staining degree,ALP activity and mineralized nodules of the cells in the S100A10 interference group were significantly reduced,and the m RNA expression levels of osteogenic related genes OCN,OPN and COL-1 were reduced.The m RNA and protein expression levels of BMP-2 signaling pathway-related factors were reduced.