The Mechanism Of Osteogenesis And Adipogenesis Of Mesenchymal Stem Cells Controlled By Niche Stiffness

Mesenchymal stem cells(MSCs)are a rich,readily accessible source of somatic stem cells.They are easily acquired,highly expanded,and efficiently differentiated into numerous lineages of specialized cells.They are used to engineer functional organs or for in situ tissue repair and regeneration.Recent research has demonstrated that the rates of trans-differentiation of MSCs to specialized cells and their engraftment in injured tissues are too low to effect substantial repair and regeneration.Therefore,it has become an important issue in regenerative medicine research to find new biological methods that can induce MSCs and clarify their regeneration and repair function and mechanism.Therefore,finding a new method that can improve the differentiation and maturity of MSCs,and clarifying its function and mechanism to promote differentiation and regeneration,has become an important subject of regenerative medicine research.Mechanical signals are a new way to regulate cell fate,in which the stiffness of the microenvironment can regulate the proliferation and differentiation of mesenchymal stem cells.Niche stiffness can induce osteogenic differentiation of bone marrow mesenchymal stem cells(BMMSCs),but the key proteins involved are not clear.Objectives:To explore the signal transduction mechanism of the stiffness of the microenvironment to guide the differentiation of mesenchymal stem cells.Methods:Isolation,purification,and identification of human bone marrow mesenchymal stem cells.Mix different concentrations of acrylamide and bisacrylamide to prepare polyacrylamide gels with different stiffness(13-16 k Pa and 62-68 k Pa).Using stiffness to induce adipogenic and osteogenic differentiation of human BMMSCs.BMMSCs were cultured on 13-16 KPa and 62-68 KPa for 24 hours.Use i TRAQ proteomics method to identify the proteins involved in stiffness-induced mechanical signal transduction,Differentially expressed proteins were selected between 62-68KPa/13-16KPa(one-tailed T-Test P-value < 0.05,fold change>1.10 or<0.91).and perform subcellular structure localization,Gene ontology(GO)enrichment analysis,Kyoto Encyclopaedia of Genes and Genomes(KEGG)enrichment analysis,protein domain enrichment analysis and network analysis.Results:1.Use the whole bone marrow culture method to obtain human bone marrow mesenchymal stem cells.The stiffness of simulated fat promotes adipogenesis of mesenchymal stem cells,and the stiffness of simulated precalcification bone promotes osteogenesis of mesenchymal stem cells.2.The protein with the highest differential expression of 62-68 k Pa /13-16 k Pa is the proline-rich protein Bst NI subfamily 3(PRB3).PRB3 is a glycoprotein rich in glycosylated proline.At present,there is no research on the involvement of PRB3 in mechanical signal transduction,and it is expected to be the object of further experimental verification.3.The differentially expressed proteins of 62-68 k Pa /13-16 k Pa are concentrated in the nucleus,cytoplasm,cell membrane and outside the cell,indicating that the position from the nucleus to the outside of the cell is involved in the signal transduction process caused by stiffness.The GO enrichment analysis of 62-68 k Pa /13-16 k Pa differentially expressed proteins suggests that in the cell composition,it is enriched to histone locus,U2-type spliceosome,transcriptionally active chromatin,prespliceosome,U2 sn RNP,Nuclear spots,spliceosome tri-sn RNP complex;in molecular function,it is enriched in nucleosome DNA binding,nucleosome binding,chromatin DNA binding,DNA binding,phosphatidylinositol diphosphate binding;in biological processes,Enriched to RNA metabolism process,gene expression,DNA as template transcription,nucleic acid as template transcription,RNA biosynthesis process,nucleic acid metabolism process,negative regulation of m RNA splicing through spliceosome,transcription of sn RNA from RNA polymerase III promoter,Regulation of RNA metabolism,negative regulation of RNA splicing,etc.The total differential protein is related to the metabolic process,suggesting that the stiffness of the microenvironment changes cell metabolism,which is consistent with the change of metabolism by mechanical stimulation.These down-regulated items are all related to gene expression regulation,mainly at the level of transcriptional regulation.It is suggested that at 62-68 k Pa,the transcriptional regulation of cells is fully down-regulated.4.Differentially expressed proteins of 62-68 k Pa/13-16 k Pa are enriched into osteoblasts for fate commitment and adipose tissue development,which are consistent with the results of mesenchymal stem cell differentiation.Osteogenic differentiation SLC26A2,SMAD5,SH3PXD2 B,BMP1,WWTR1,FAT4,PENK;Adipogenic differentiation TBL1XR1,SH3PXD2 B,SPG20.The key genes DDX49,SNRPG,SF3A2,RRP9,LSM8,PTBP1,SRSF4,ELL,UPF3 B,and KLHL22 predicted by the protein interaction network are currently not related to mechanical stimulation.It may play a key role in the signal transduction that rigidity guides the differentiation of mesenchymal stem cells.Conclusions:1.Microenvironment stiffness controls the fate of mesenchymal stem cells.Hard environment induces osteogenic differentiation of mesenchymal stem cells,and soft environment induces adipogenic differentiation of mesenchymal stem cells.2.Proteomic data support that microenvironment stiffness controls mesenchymal stem cell differentiation,transcription regulation,cell metabolism,Hippo pathway and calcium related pathway proteins are expected to become new molecular mechanisms of adipogenic differentiation of mesenchymal stem cells.