MicroRNA124 And MicroRNA21-5p Promote Mesenchymal Stem Cell Migration,Proliferation And Differentiation Into The Nerve In Vitro And In Vivo

[OBJECTIVE] Spinal cord injury(SCI)is one of the most complex and variable pathological processes in central nervous system injury.Its treatment has always been a research hotspot and a difficult problem.Studies have confirmed that the transplantation of mesenchymal stem cells(MSCs)can promote the repair of SCI tissue in animal models,but its function is very limited.MSCs have low immunogenicity,multi-differentiation potential and strong self-renewal ability,and can be used to treat a variety of diseases.Their functions are regulated by various cytokines,transcription factors,signaling pathways,and micro RNAs(mi RNAs).Both mi R124 and mi R21-5p are closely related to the function of nerve cells.This study explored the role of mi R124 and mi R21-5p in the migration,proliferation,and differentiation of MSCs into neural cells,and further explored the role of MSCs transplantation in the repair of SCI.[METHODS] MSCs were isolated and cultured from bone marrow of SD rats using whole bone marrow adherent culture method and expanded in vitro to P3-P5 generation for further experiments.The MSCs were divided into four groups,the first is the normal control group(NC-MSCs).The mi RNA-mimics control,mi R124-mimics,and mi R21-5p-mimics were transfected into MSCs using Lipofectamine 2000 and they were defined as transfected mimics control group(MC-MSCs),mi R124-MSCs group and mi R21-5p-MSCs group.The cell migration ability of each group was examined by wound healing assay.MSCs were cultured with EDU-containing medium and the proliferation ability of each group of cells was detected by immunofluorescence staining.The induced culture medium containing beta-mercaptoethanol(β-ME)and butylhydroxyanisole(BHA)was used to induce differentiation of MSCs into neural cells,and neuronal markers,TUJ-1 and NEUN,of the induced cells were detected by immunofluorescence staining.Rat spinal cord contusion model was established by using aneurysm clamp method.MSCs were labeled with CM-Dil or EDU in vitro.MSCs were transplanted into rat by tail vein injection or lumbar puncture injection.The spinal cord tissue of rats was frozen and sectioned for immunofluorescence staining.The number of cells after transplantation and differentiation into neurons were observed.[RESULTS] Through the whole bone marrow adherent culture method,we can easily obtain a large number of MSCs.Through the induction of β-ME and BHA,MSCs can be successfully induced to differentiate into neural cells in vitro.Using Lipofectamine 2000 in combination with mi R124-mimics or mi R21-5p-mimics,MSCs highly expressing mi R124 or mi R21-5p can be obtained.Overexpression of mi R124 can promote the migration and differentiation of MSCs into neural cells.Overexpression of mi R21-5p can promote the proliferation of MSCs and differentiate into neural cells.The rat spinal cord model prepared by the clamp method has the advantages of simple operation,exact damage,quantification,high repeatability,and low postoperative mortality.CM-Dil can efficiently label MSCs in vitro and in vivo.Through the tail vein injection and lumbar puncture injection,MSCs can be successfully transplanted into the SCI site,and lumbar puncture transplantation of MSCs can gain more transplanted cells in the spinal cord injury.At the same time,transplanted cells can differentiate into GFAP-positive cells.[CONCLUSION] The MSCs obtained by the whole bone marrow adherent culture method can be differentiated into neuron-like cells in vitro.Mi R124 can increase the ability of MSCs to migrate and differentiate into neural cells.Mi R21-5p can increase the proliferation of MSCs and their ability to differentiate into neural cells.CM-Dil can efficiently label MSCs in vitro and in vivo.Compared with tail vein injection,lumbar puncture transplantation of MSCs can obtain more transplanted cells at the site of spinal cord injury,and the transplanted cells can successfully migrate to the spinal cord injury site and differentiate into nerve tissue,which is of great significance for further repair of spinal cord injury.