FGF-2 Transfection And Induction Of Rat Bone Marrow Mesenchymal Stem Cells Differentiation Into Cardiomyocyte-like Cells

Since adult cardiomyocytes are terminally differentiated cells,myocardial damage caused by ischemic heart disease cannot be repaired by regeneration of autologous cardiomyocytes.Therefore,how to restore infarcted myocardial tissue to restore its original function is the key to treat ischemic heart disease.Bone marrow mesenchymal stem cells(BMSCs)are common "seed cells".BMSCs can be differentiated into multiple directions under the certain conditions,such as fat,bone,muscle,nerve,heart muscle,endothelium,etc.Bone marrow mesenchymal stem cells can also be used as root cells to delay aging and repair.Tissues and organs damaged by disease.Bone marrow mesenchymal stem cells,which can induce to differentiate into cardiomyocyte-like cells,were implanted into damaged myocardial tissue and have a certain effect on repairing myocardial damage.Fibroblast growth factor-2(FGF-2)is a multifunctional growth factor widely distributed in various tissues in the body.It stimulates the migration,proliferation and differentiation of various stem cells,promotes the formation of new blood vessels and protects the heart.With the continuous development of transgenic technology,the transformation of stem cells by means of gene transfection has become intense in the field of stem cell research.In gene modification,lentiviral vector is the most widely used gene vector.Compared with the vectors such as plasmid and adenovirus,it has the advantages of high transfection efficiency,low cytotoxicity and long expression time of the target gene.In this study,FGF-2 gene was transfected into BMSCs by constructing lentiviral vector,and transfected BMSCs overexpressed FGF-2 gene.To explore the role of FGF-2 in the differentiation of BMSCs into cardiomyocyte-like cells,and to compare whether gene transfection is superior to direct drug induction.It is expected to enhance the differentiation efficiency of cells and provide an excellent cell source for clinical treatment of ischemic heart disease by cardiomyocyte transplantation.To investigate the effect of FGF-2 lentivirus transfection on the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells,the direct induction of FGF-2 was better.In this experimental study,BMSCs were isolated and cultured under aseptic conditions in vitro by whole bone marrow adherence method.Morphological observation was performed on the amplified and purified cells.BMSC began to adhere after 6 hours and adhered after 24 hours.It is round and has strong refraction;after 2 days,most of the cells were adherent,most of them are short spindle-shaped,and a small number of cells are irregular;after 4 days,the cell volume begins to increase,and the spherical cells gradually Decrease;after 7 days,the cell volume gradually became larger and began to spread to the surroundings,and the cells were arranged in a substantially parallel arrangement.The fourth generation of BMSCs was identified by flow cytometry.The results showed that the positive expression rate of CD45 was 4.6%;the positive expression rate of CD29 was 98.2%,and the positive expression rate of CD90 was 91.8%,which was consistent with the characteristics of BMSCs.The fourth generation of cultured cells was identified as purified BMSCs.Before induction,the optimal induction concentration of the drug and the MOI value of the optimal virus for lentivirus transfection were screened.The pre-experimental study showed that the optimal induction concentration of FGF-2 might be 1 ng/ml,Lenti-FGF-2-GFP.The optimal transfection concentration of lentivirus was MOI=150 and the optimal transfection concentration of Lenti-control-GFP lentivirus was MOI=100.The inducer was added to the cells cultured to the second passage,and the cells were exchanged after 72 hours of induction,and replaced with IMDM medium containing no inducer to continue the culture.According to the different inducing agents,they were divided into the following four groups: group A(BMSCs blank control group),group B(FGF-2 induction group),group C(Lenti-FGF-2-GFP lentivirus transfection group)and the group D(Lenti-control-GFP lentiviral transfer)were determined by pre-experiment to determine the optimal cell-inducing density,optimal drug-induced concentration and optimal induction time.Real-time PCR was used to detect the expression of early myocardial transcription factors GATA-4 and Nkx2.5.The results showed that GATA-4 and Nkx2.5 were expressed in all groups of cells.As a reference control,GAPDH was used to determine the expression of GATA-4 and Nkx2.5 genes by relative quantitative analysis.The results showed that compared with the experimental control group,the expression of GATA-4 and Nkx2.5 genes was the strongest after induction of 4 weeks in each induction group.Among them,the relative expression levels of GATA-4 and Nkx2.5 genes in FGF-2 transfection group were highest at all time points.The expressions of cTnI,cTnT,Cx43 and Desmin were detected by immunocytochemical staining and immunofluorescence staining.After 4 weeks of induction,cTnI,cTnT,Cx43 and Desmin were positively expressed in the cytoplasm of cells.Statistical analysis showed that the integrated optical density(IOD)values of the markers in the FGF-2 transfection group were the strongest.Cx43 and cTnI were weakly positive or negative in the experimental control group.There was a significant difference in the positive expression of each marker in each induction group and the experimental control group.The expressions of Tm and Desmin were detected by Western blot.The results showed that Tm and Desmin were expressed in each experimental group after 4W induction,and the positive expression rate of Desmin in group C was higher than that in other experimental groups,and the expressions of Tm in group B and C were higher than those in group A and D.The results of transmission electron microscopy showed that the nucleus of BMSCs transfected with fgf-2 lentivirus was oval and located in the center of the cell,and there were myofilaments,rough endoplasmic reticulum,mitochondria,ribosome and other organelles in the cytoplasm.It accords with the structural characteristics of myocardial cells during development.This experiment shows that:(1)FGF-2 can transfect and induce differentiation of BMSCs into cardiomyocyte-like cells.(2)Lentivirus-mediated FGF-2 transfection induces the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells better than FGF-2 direct induction.