| At present,cardiovascular diseases still threaten human health.Among them,myocardial infarction(MI)is one of the most common and most serious type.MI is mainly due to the irreversible loss of myocardial cells,adverse remodeling of the ventricle,and cardiac dysfunction.Although important progress has been made by controlling some risk factors,combined with drug therapy and stent intervention therapy,the main purpose is still to alleviate and control the disease,instead of directly repairing and regenerating the damaged myocardium,and then restoring myocardial function.The continuous development of MI eventually leads to heart failure.Although heart transplantation is used as the only fundamental treatment,it cannot meet clinical needs due to the limited donors and the uncontrollable immune response.In order to alleviate the situaion,some scholars have found that transplanting exogenous stem cells to the injured site of myocardium can reduce the speed of infarction and repair the infarcted area.Therefore,in tissue engineering,stem cells become ideal seed cells for myocardial repair and regeneration.Bone marrow mesenchymal stem cells(BMSCs)are one of the most common stem cells.Due to the natural biological characteristics of BMSCs,they have a certain effect on the treatment of MI,such as the secretion of some mediators and growth factors.The effects of angiogenesis,immunosuppression,anti-apoptosis,inhibition of inflammation,anti-fibrosis,etc.,promote stem cells to become important seed cells for the treatment of MI.When BMSCs treat MI,the number of BMSCs is extremely high,and differentiated cardiomyocytes are required to be similar to natural cardiomyocytes.Therefore,there are strict requirements on the factors and methods for inducing BMSCs to differentiate into cardiomyocytes.5-azacytidine(5-Aza)has been recognized as the most important cardiogenic inducer as early as the twentieth century.It can effectively induce BMSCs to differentiate towards the myocardium due to its important demethylation Therefore,5-Aza has always been a research hotspot.Among the cytokines in biological factors,bone morphogenetic protein 2(BMP-2)belongs to the transforming growth factor?family,which has a regulatory effect on the proliferation,survival,differentiation and apoptosis of a variety of cells In addition to being able to differentiate into osteoblasts,it is a key upstream signal for myocardial development during embryonic development.Many studies have found that although BMSCs can be induced to differentiate toward the myocardium in vitro,there are also problems such as insufficient proliferation of cardiomyocytes.Therefore,in addition to the combined induction of multiple factors,the advancement of gene therapy technology has played a role in promoting the application of BMSCs.Currently,viral vectors are widely used in gene therapy,among which lentiviral vectors are the most commonly used vectors.Compared with other vectors,they have stable and long-term expression,higher viral titer,lower cytotoxicity,and host types.Extensive and other characteristics.This experiment mainly constructs a lentiviral vector,transfects BMP-2gene into BMSCs through overexpression,and explores whether the efficiency of overexpression of BMP-2 gene to induce BMSCs to differentiate into cardiomyocytes is higher than that of direct induction of BMP-2,and at the same time explore overexpression of BMP-2 gene combined with 5-Aza in inducing BMSCs to differentiate into cardiomyocytes is higher than the efficiency of other independent induction.In this experiment,the whole bone marrow adherence method was used to isolate and culture BMSCs,the surface antigen was identified by flow cytometry,and the optimal concentration of BMSCs induced by BMP-2 in rats after transfection was screened.Immunocytochemistry and Western blot technology Detect the positive expression of related specific proteins,detect the expression efficiency of related genes by real-time fluorescent quantitative PCR(RT-q PCR);observe the ultrastructure of differentiated cardiomyocytes with transmission electron microscope(TEM).Observe the morphology of the cells under a microscope:after 72 hours of culture,the cells are basically adherent,one part is round and refracts bright light,and the other part is stretched and has a short spindle shape;after culturing to 1w,the cells show diversified morphology,including spindle and diamond shapes,Polygonal;after culturing to 4w,the cells are closely connected,have a directional arrangement,and are radial or swirling.Flow cytometry was used to identify the surface antigens of the third generation of BMSCs.The results showed that the positive expression rates of CD45,CD29,and CD90 were 1.4%,93.3%,and 95.1%,respectively.The results showed that the cells cultured to the third generation were purified BMSCs.Before grouping,we first screened the optimal MOI value of BMSCs induced by overexpression of BMP-2 gene by constructing lentiviral vector.Preliminary experimental results showed that the optimal MOI value of Lenti-BMP-2-GFP lentiviral transfection group was 25.The optimal MOI value of the Lenti-control-GFP lentivirus transfection group was 50.According to the different inducers,the cells cultured to the third passage are grouped into experiments:G1(blank control group),G2(BMP-2group),G3(Lenti-BMP-2-GFP lentivirus transfection group),G4(Lenti-BMP-2-GFP lentiviral transfection+5-Aza group),G5(Lenti-control-GFP lentiviral transfection group),the final concentration of BMP-2 is 200ng·L-1,the final 5-Aza The concentration is 10?mol·L-1.The results of immunocytochemistry and Western blot technology showed that after the cells were cultured for 4 weeks,each induced group had cardiac troponin T(c Tn T),Desmin and tropomyosin(TPM)and connexin 43(Cx43)positive expression,the results showed that:c Tn T,Desmin,TPM,Cx43 positive expression in each group,and the Lenti-BMP-2-GFP+5-Aza group had the highest positive expression rate,and the difference was statistically significant(P<0.05).Real-time fluorescent quantitative PCR(RT-q PCR)method was used to detect the expression of myocardial early transcription factors GATA-4,Nkx2.5 and MEF2c.The induction time of each induction group was 1w,2w and 4w,and the results showed:GATA-4 and Nkx2.5 genes were expressed at 1w in each induction group,and then the expression continued to increase.The expression efficiency was the strongest at 4w.The expression of Lenti-BMP-2-GFP lentiviral transfection+5-Aza group was expressed at 4w.Compared with the expression of 1w and 2w,the difference was statistically significant(P<0.05).MEF2c gene began to be expressed at 1w in each induction group,and the expression efficiency was highest at 2w.The expression efficiency gradually weakened after 2w,Lenti-BMP-2-GFP lentivirus transfection+5-Aza group had the highest expression efficiency at 2w,and the difference was statistically significant compared with 1w and 4w(P<0.05).Transmission electron microscopy(TEM)observation results showed that the BMSCs cells induced by Lenti-BMP-2-GFP lentivirus transfection+5-Aza group showed myofilament,mitochondria,rough endoplasmic reticulum,ribosomes and other organelles in the cytoplasm,Has an ultrastructure similar to cardiomyocytes.Explanation of experimental results:(1)The efficiency of inducing BMSCs to differentiate into cardiomyocytes by constructing a lentiviral vector to overexpress BMP-2 is higher than that of direct induction by BMP-2.(2)The efficiency of overexpression of BMP-2 gene combined with5-Aza to induce BMSCs to differentiate into cardiomyocytes is higher than that of direct induction of overexpression of BMP-2. |
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