MiR-130b Is Involved In The Balance Between Osteogenic And Adipogenic Differentiation Of Human Bone Marrow-derived Mesenchymal Stem Cells

Objective:Osteoporosis(Osteoporosis,OP)is a kind of metabolic and systemic skeletal disease with decreased bone mass,bone microstructure and bone fragility damage increased as the major pathological features,resulting in reduced bone strength so as to induce different degrees of fracture.The main clinical manifestations are pathological fracture and low back pain,and most of them occur in middle-aged and old people.With the acceleration of the aging of the world population,senile osteoporosis and its fracture are becoming more and more concerned around the world.The characteristics of senile osteoporosis are osteopenia and fat accumulation in bone marrow.This imbalance arises from the imbalance between osteogenic and adipogenic differentiation of Bone Marrow Mesenchymal stem cells(BMSCs).Bone marrow mesenchymal stem cells(BMSCs)can play a multidirectional and self renewing function.They are widely distributed in bone marrow tissues of human beings and other organisms.It can be differentiated into osteoblasts,adipocytes and chondrocytes under certain conditions.BMSCs,as a progenitor of osteoblasts and adipocytes,is the root cause of the imbalance of bone formation and fat formation by the disequilibrium of osteogenesis and lipid differentiation.There is a reverse change in the differentiation of BMSCs into osteoblasts and adipocytes.This conclusion is very important for the study of the pathogenesis of osteoporosis.The study found that miRNA participates in the differentiation and regulation of BMSCs and determines the differentiation direction of stem cells.Our previous study found that miR-130 b showed a high expression trend in BMSCs osteogenic differentiation,and showed a low expression in the process of adipogenic differentiation.This suggests that miR-130 b is involved in two processes of osteogenesis and adipogenesis.But what is the specific role of miR-130 b in the process of BMSCs osteogenesis and lipid differentiation? Does miR-130 b play a role in regulating BMSCs osteogenesis and lipid differentiation? These are not yet clear.Therefore,this study based on BMSCs system of adipogenic differentiation and osteogenic,by upregulating miR-130 b expression,to study the effects of osteogenic and adipogenic differentiation ability of BMSCs,the further study of miR-130 b in bone marrow mesenchymal stem and adipogenic differentiation of cells into balance.Methods:(1)human bone marrow mesenchymal stem cells(BMSCs)were artificially resuscitation and subculture,and cell surface markers(CD29,CD44,CD90,CD105,CD34 and CD45)were analyzed by flow cytometry.(2)osteogenesis,lipid induced differentiation and cytological identification of human bone marrow mesenchymal stem cells(BMSCs).Osteogenic differentiation culture system: H-DMEM medium(containing10mmol/L sodium glycerophosphate and 0.1μmol/Ldexamethasone,10%FBS,50 mol/L ascorbic acid phosphate);adipogenic differentiation culture system: H-DMEM medium(containing 10%FBS,1μmol/Ldexamethasone,200μmol/L Indometacin,0.5mmol/LIBMX,10μg/ml indomethacin).(3)lentivirus vectors expressing miR-130 b and NC were constructed respectively,and BMSCs cells were infected by different titer viruses.(4)after increasing the expression level of miR-130 b and NC,BMSCs was induced to differentiate into osteoblasts and adipocytes,and related identification analysis was performed to detect the changes of osteogenic and adipogenic differentiation ability.Results:(1)the cells passage from the passage to recovery showed that the expression of CD29,CD44,CD90 and CD105 was positive,and the expression of CD34 and CD45 was negative,which was consistent with the characteristics of human bone marrow mesenchymal stem cells(BMSCs).(2)after 7 days of osteogenic induction,the cells were gradually synthesized into a paving stone by inverted microscope.As time went on,cells began to accumulate and form multiple nodules.After 14 days of alkaline phosphatase staining,brown nodular mineralized nodules were found,and small nodular mineralized nodules were formed after 21 days of culture.(3)after adipogenic were observed from the initial shape of long spindle to polygonal or circular transformation induced by inverted microscope after 7 days,after oil red staining of the cytoplasm appeared small lipid droplets,cell disorder,lipid levels increased,cell volume change,differentiation after 2 weeks showed large amounts of lipid droplets form.(4)the miR-130 b and NC lentivirus vectors were constructed respectively,BMSCs cells were transfected successfully,and the BMSCs cell lines expressing miR-130 b and the control group cell lines were established.(5)expression of BMSCs cells miR-130 b cells compared with the control group: after osteogenic induction and differentiation culture showed its osteogenic characteristics stronger alkaline phosphatase activity increased significantly,the expression of Western Blot than the display detection of osteogenic related gene RUNX2 in the control group increased;after adipogenic differentiation culture visible after adipogenic properties weakened,oil red staining showed that the number of lipid and fat cells decreased,Western Blot showed that the expression of genes related to lipid CEBP Alpha was lower than the control group.Conclusion:(1)human bone marrow mesenchymal stem cells(BMSCs)have good biological activity after resuscitation and subculture,and can be used as seed cells.(2)human bone marrow mesenchymal stem cells(BMSCs)can differentiate into osteoblasts and adipocytes under the action of different inducers.(3)the BMSCs cell line was successfully established to increase the expression level of miR-130 b.(4)up regulation of the expression level of miR-130 b can promote the osteogenic differentiation of BMSCs,and inhibit the ability of lipid differentiation.