The Mechanism Of MiR-125b Reg?lates Osteogenesis In Human Bone Marrow Mesenchymal Stem Cells

BackgroundWith the aging of the domestic pop?lation,many diseases related to aging have caught extensive attention in medical science.As a high incidence of disease in the elderly pop?lation,osteoporosis,especially the severe osteoporosis is the high fracture risk.Meanwhile,such fractures tend to delay in recovering after occurrence.It is a normal orthopedic disease which seriously affects the health and quality of life of elderly.The WHO has defined the osteoporosis,diabetes,and cardiovasc?lar disease as "three killers" which affect the health of the elderly.As an important a precursor cells of osteoblasts,human bone marrow mesenchymal stem cells(hBMSCs)plays an important role in bone formation and osteogenetic differentiation.The study of the mechanism of ectomesenchymal stem cells for osteogenesis helps to better understand the pathological process of a variety of bone disease and look for new ideas for the treatment of such diseases.In addition,the hBMSCs has strong ability of proliferation and m?lti-directional differentiation potential,and it is the most important source of seed cells for bone tissue engineering,and it also has extensive application prospect in the field of stem cell transplantation.Micro RNA(miRNAs)is a non-coding single small rnas which widely exists in eukaryotic cells.It can combine with 3' non-translation section of target genes,then reg?late the expression of genes.It plays an important role in many of the basic physiological and pathological process.In recent years,more and more studies suggest that miRNAs plays an important role in reg?lation of dynamic balance of osteoblast and osteoclast,and it is directly involved in a variety of bone diseases such as osteoporosis,arthritis and other diseases of the occurrence and development process.Studies have found that miR-125 b decreased in the process of osteogenesis,but the specific reg?latory mechanism is not f?lly clear.This study selected miR-125 b as the research object,hoping to further enrich the theory system of directional differentiation of stem cell development through the miR-125 b reg?lation between human bone marrow mesenchymal stem cells in the osteogenetic differentiation mechanism research,as well as deepen the understanding of bone molec?lar mechanism of occurrence,development,for the prevention and treatment of osteoporosis and bone fracture treatment,and looking for a new treatment of bone defect and providing theoretical support.Methods1.Isolation,c?lture and identification of human bone marrow mesenchymal stem cell.Isolated and c?ltured hBMSCs from the healthy ad?lt volunteers used the method of density gradient centrifugation,,and got stable source of stem cells by passaging,c?lturing and purifying.We also observed adherent cells in the process of c?lture,detected the proliferation activity by MTT.Qualifid the cell identification properties by cell surface antigens markers was detected to qualify the cell identification with the flow cytometry,and identified the capacity of the m?lti-directional differentiation of hBMSCs by detected the osteogenesis,adipogenic ability,chondrogenic ability.2.The effects of miR-125 b on hBMSCs osteogenesis differentiation.Lentiviral vector transfection method was used to transfect hBMSCs.Firstly,builded the synthetic miR-125b-pre and MiR-125 b inhibitor nucleic acid sequence into the Lentiviral vector and then transfecting hBMSCs.Then observed and tested the transfection efficiency and proliferation of cells,detected the express of the MiR-125-b in different groups after transfection with the Real Time RT-PCR technology.Finally,the mRNA and protein expression of ALP,Runx2,OSX,the important factors in the osteogenetic differentiation of mesenchymal stem cells were detected using Real Time RT-PCR and Western Blot.alkaline phosphatase staining and alizarin red staining of cells were used after transfection,observed grade of cell color stain to Identify osteogenetic differentiation ability.3.prediction and validation of the target of miR-125 b in reg?lation hBMSCs.First,used bioinformatics methods,the target gene prediction tools including Target Scan,miRanda and Pic Tar.predicted and screened possible target gene of miR-25-b,selected the potential target BMPR1 b as the research object.Then,constructed the luciferase report BMPR1 b vector,used the method of dual luciferase report to predict target genes for preliminary validation.Finally,the hBMSCs was transfected miR-125b-inhibitor and miR-125 b respectively,the mRNA and protein expression of BMPR1 b were detected during miR-125 – b changed by expression changes Real Time RT-PCR and Western Blot..4.The mechanism of inhibition in hBMSCs osteogenesis differentiation by MiR-125-b.The first to detect the expression of BMPR1 b during osteogenesis differentiation by Real Time RT-PCR and Western Blot testing.Then,using RNA inhibitor technology,transfected BMPR1 b siRNA vectorion into hBMSCs,the mRNA and protein expression of Runx2,OSX and OCN were detected by Real Time RT-PCR and Western Blot.alkaline phosphatase staining and alizarin red staining of cells were used after transfection,observed grade of cell color stain to Identify osteogenetic differentiation ability.5.The study of reg?lation of osteogenetic differentiation of by MiR-125-b in vivoFirst of all,established a nude mice model of the femoral bone defect in situ.Then,transfected MiR-125-b inhibitor to hBMSCs,combinated hBMSCs to DBM after hBMSCs had induced osteogenetic c?lture,transplanted DBM into nude mice by surgery,micro CT was used for observation of new bone formation,tissue section,HE staining and Masson staining,was used to Evaluate the osteogenesis ability.Res?lts1.This study used density gradient centrifugation to isolate and c?lture of hBMSCs,hBMSCs cells has normal morphology,good proliferation activity.The percentage of HBMSCs which express positive surface antigen maker CD73,CD90 and CD105 were over 95%,while The percentage of HBMSCs which express negative antigen maker CD34,CD45,CD14,CD19,HLA-DR were below 5%,conformed HBMSCs standard.In addition,the c?ltivation of hBMSCs have m?lti-directional differentiation capacity,including osteogenesis,adipogenic ability,chondrogenic ability.2.In the process of hBMSCs to the osteogenetic differentiation,the expression of miR-125 b was in low level.Lentiviral vector of hBMSCs transfection was succeeded,had no influence on cell proliferation.In the process of hBMSCs osteogenesis differentiation,up-expression of MiR-125-b can inhibited osteogenesis differentiation.On the contrary,the down-expression of MiR-125-b co?ld promote osteogenesis differentiation.3.Bioinformatics predicted BMPR1 b gene may be target of miR-125.Dual luciferase report experiments have established that MiR-125-b and BMPR1 b had binding sites in 3 'UTR part base sequence.up-expression of MiR-125-b inhibited mRNA and protein expression of BMPR1 b.Instead,down-expression increased mRNA and protein expression of BMPR1 b.4.BMPR1 b got high expression during hBMSCs osteogenetic differentiation.After silence BMPR1 b gene,the ability of hBMSCs osteogenesis was decreased.co-transfected MiR-125-b inhibitor and si-BMPR1 b to hBMSCs,the ability of hBMSCs osteogenesis also was restrained.5.The study of osteogenesis reg?lation by MiR-125-b in vivo had found that the hBMSCs which transfected miR-125b-inhibitor had better osteogenesis adifferentiation ability micro CT and biopsy staining was used.Conclusion1.The hBMSCs which were used in our study had good proliferation ability,force is good,conformed HBMSCs morphology standard.between general of mesenchymal stem cells.hBMSCs,induced by different conditions,to osteogenesis,into fat and differentiation into cartilage cells the c?ltivation of hBMSCs have m?lti-directional differentiation capacity,including osteogenesis,adipogenic ability,chondrogenic ability.It comformed we had stable and reliable source of hBMSCs.2.MiR-125-b can negative reg?lation hBMSCs osteogenetic differentiation.3.MiR-125-b targeted part 3 'UTR bases of BMPR1 b to inhibitthe expression of BMPR1 b.4.MiR-125-b inhibited the expression of its target genes BMPR1 b,thereby inhibiting hBMSCs osteogenetic differentiation.5.After down reg?lated miR-125 b expression,the osteogenic differentiation ability of hBMSCs co?ld be enhanced in vivo.