The Mechanism And Effect Of Mesenchymal Stem Cells-Endothelial Progenitor Cells Promot The Bone Regeneration And Repairing

Objective:To investigate the effect of endothelial cells-bone marrow mesenchymal stem cells co-culture system on bone regeneration and its molecular mechanism in vitro.Methods:1.Bone marrow mesenchymal stem cells(BMSC)and endothelial cells were isolated from the bone marrow.Two kinds of cell morphology were observed under the microscope in vitro.Differentiation of BMSC was induced by adipogenic differentiation and osteogenic differentiation induction medium.Differentiation ability of BMSC was detected by oil red O staining and alizarin red S staining.Surface molecular markers of BMSC and EPCs were detected by flow cytometry and immunofluorescence.The angiogenesis ability of EPC cells was detected by in vitro culture tube test.EPCs marker gene expression was detected by RT-qPCR.The expression levels of osteoblast related genes were detected by Western blot.2.EPC+BMSC co-culture system was constructed in vitro.RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of bone-forming related genes.Alizarin red staining was used to detect the formation of calcified nodules in BMSC cells.The proliferation activity of BMSC was detected by CCK-8.ALP activity assay kit quantifies ALP activity;ALP expression and distribution were detected by immunohistochemistry.Bone formation of BMSC was observed by SEM.3.CCK-8 experiment was used to detect the proliferation activity of EPC.The expression of endothelial marker protein in EPC was detected by immunofluorescence;Rt-qpcr and Westernblot were used to analyze the expression of endothelial cell related gene mRNA,and Transwell was used to detect the migration ability of EPC cells.EPC angiogenesis was detected by in vitro culture.4.Prepare conditioned medium supernatant(CM-S)of EPC and BMSC?Treat BMSC with EPC-CM-S,Treat EPC with MSC-CM-S;BMSC is reprocessed with EPC-CMS from EPC source after BMSC-CM-S treatment;The secretion levels of BMP2 and VEGF in the cells of each culture group and the co-culture system were detected by ELISA.RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of bone-forming related genes.The ALP level of BMSC microslide was detected by immunohistochemistry.The expression of Runx2 and Osterix(Osx)related transcription factors of osteogenic differentiation in BMSC cells was detected by immunofluorescence assay.5.EPC-exosomes were cultured and isolated,the morphology of exosomes was observed by transmission electron microscope,and the expression of exosome surface markers was detected by Western blotting.EPC-exosomes were co-cultured with BMSC cells to explore the effect of miR-212-3p from EPC exosomes on the osteogenic differentiation of BMSC through smad-7.CCK-8 and Transwell was used to test proliferation activity and migration ability of BMSC cells respectively.ALP activity was detected by ALP kit,and expression of osteogenic differentiation related genes was detected by RT-qPCR and Westemblot respectively.Alizarin red staining was used to detect the formation of calcified nodules in BMSC.The targeting relationship between miR-212-3p and Smad-7 was verified by the dual-luciferase reporter gene assay.6.lncRNA-seq chip was used to detect the differentially expressed lncRNAs in EPC cells of co-culture system.CCK-8 assay was used to detect the proliferation activity of EPC cells.Transwell assay was used to detect the migration ability of EPC cells.The expression levels of marker genes and proteins in endothelial cells were detected by RT-qPCR and Western blot.Angiogenesis of EPC cells was tested by in vitro tube formation.Results:1.EPC and BMSC cells were successfully isolated from rat bone marrow.2.Compared with cultured BMSC cells alone,EPC+BMSC co-culture system significantly promoted the proliferation activity,ALP activity,osteogenic related gene expression level and calcium nodule formation of BMSC cells.3.Compared with EPC cells cultured alone,EPC+BMSC co-culture system significantly promoted the proliferation activity,migration ability,expression level of endothelial cell marker protein,and in vitro tube formation ability of EPC cells.4.EPCs cultured alone did not secreted VEGF-A and BMP-2,BMSCs cultured alone did not secreted BMP-2,but secreted VEGF-A.Both VEGF-A and BMP-2 in the co-culture system were highly secreted.EPC condition supernatant does not initiate and promote osteogenic differentiation of BMSC.EPC cells activated by BMSC promote osteogenic differentiation of BMSC by secreting BMP-2.5.miR-212-3p was abnormally expressed in BMSC cells treated with EPC-exosome,Knocking down of miR-212-3p significantly reduced the osteogenic differentiation level of BMSC.EPCs exosome miR-212-3p promotes BMSC cell proliferation,migration,and osteogenic differentiation by down-regulating SMAD7.6.lncRNA MALAT1,lncRNA MEG3,lncRNA ATB and IncRNA HULC were highly expressed in EPC cells of co-culture system,and the up-regulated multiple of IncRNA MALAT1 was the highest.Knocking down LncRNA MALAT1 significantly reduced the proliferation,migration,endothelial cell differentiation and ductal ability of EPC cells in the low co-culture system.The co-culture system significantly promoted the up-regulation of IncRNA MALAT1 expression in EPC cells,and promoted the proliferation,migration,endothelial cell differentiation and vascular formation of EPC cells by reducing the negative regulatory effect of mir-124-3p on SOX7.Conclusion(s):1.Co-culture of EPC and MSC can significantly promote osteogenic differentiation and vascular endothelial cell differentiation during bone regeneration.2.In the co-culture system,BMSCS secreted vegf-a to stimulate and activate EPC cells,and the activated EPC cells secreted MBP2 to promote osteogenic differentiation of BMSCS.This is a "BMSC-EPC-BMSC-feedback loop" mode of regulation.3.The interaction or mutual regulation between EPC and BMEC in the co-culture system is mainly through two ways:Direct secretion of key cytokines to regulate osteogenesis or angiogenesis;One cell transfers nucleic acid or protein regulators by the form of exosome vesicles that affect the differentiation or biological behavior of another cell.4.miR-212-3p,Smad-7,LncRNA MALAT1,miR-124-3p and SOX7 may be potential markers of bone regeneration and angiogenesis.