| BACKGROUND:Cardiovascular disease(CVD),as one of the major diseases in the world that harm the life and health of human beings,especially the middle-aged and the elderly,has attracted much attention from doctors and scholars because of its extremely high incidence and the trend of getting younger in recent years.As an important member of cardiovascular diseases,acute myocardial infarction(AMI)has become a hot and difficult topic in scientific experiment and clinical treatment.Mature cardiomyocytes are regarded as terminal differentiated cells.When myocardial infarction occurs,the cardiomyocytes are damaged to different degrees and even suffer necrosis or apoptosis,resulting in loss of function.Clinical treatment methods for AMI mainly include drug therapy,interventional therapy,etc,all of which are aimed at the early stage of myocardial infarction to save the damaged but not yet apoptotic myocardial cells and their functions,but for the myocardial cells that have been necrotic or apoptotic,there is no recovery.Cardiomyocyte necrosis,functional cardiomyocytes are replaced by nonfunctional fibroblasts to form scar tissue,ventricular remodeling,cardiac function decline,and eventually lead to heart failure,resulting in decreased quality of life of patients,which seriously threatening the life of patients.Due to the irreversibility of myocardial cell injury,the lack of myocardial cells is the fundamental problem in the treatment of myocardial infarction.With the progress of science and technology and the development of cell bioengineering,stem cell replacement therapy has become a promising new treatment method for myocardial infarction.Stem cells have the ability of self-replication and multidirectional differentiation,which causes the treatment of myocardial infarction by stem cell transplantation to become a research hotspot.Several basic and clinical trials have shown that stem cell transplantation can repair damaged cardiomyocytes,promote angiogenesis,reduce infarct size,and promote recovery of heart function.Since 2003,the US FDA has been the first to approve autologous bone marrow stem cell transplantation for the treatment of myocardial infarction and other diseases.Since then,research on the treatment of stem cells has been carried out on a global scale,among which bone marrow mesenchymal stem cells are the most widely used and mature.BMSCs compared with stem cells,such as embryonic stem cells have accessible,easy separation,in vitro culture and amplification has stability,no immune rejection problem,does not involve in medical ethical questions,with multi-directional differentiation potential,under some appropriate conditions using proper inducers that can differentiate into bone cells,cartilage cells,nerve cells,fat cells,endothelial cells,stem cells and smooth muscle cells,myocardial cells,etc.These advantages make BMSCs an important seed cell for stem cell transplantation in the treatment of cardiovascular diseases such as myocardial infarction.BMSCs can be differentiated into cardiomyocytes by co-culture with cardiomyocytes,gene modification and drug induction,but the differentiation rate and safety of BMSCs need to be improved.Puerarin is an important component of pueraria root and able to affect the differentiation of bone marrow mesenchymal stem cells.Puerarin and its various preparations have been widely used in the treatment of cardiovascular diseases.Puerarin,as an effective component of traditional Chinese medicine,has the advantages of smaller toxic and side effects and safer pesticide effect compared with western medicine.Clinical promotion value is significant.At present,it has been found that puerarin can not only protect damaged cardiomyocytes and the cardiovascular system,but also induce embryonic stem cells to differentiate into cardiomyoid cells in vitro and improve the differentiation efficiency of ventricular cells.PURPOSE:In this study,bone marrow mesenchymal stem cells were induced to differentiate into cardiomyoid cells,directional differentiation from the aspects of morphology and molecular biology to explore between puerarin on bone marrow mesenchymal stem cells for the ability of myocardial cells,the optimal concentration of screening through its differentiation increase the rate of differentiation,for cardiovascular diseases such as myocardial infarction,which provides the theoretical foundation for stem cell transplantation therapy.METHODS AND RESULTS:To investigate the feasibility of puerarin inducing BMSCs to differentiate into cardiomyoid cells,BMSCs from limb bones of SD rats were isolated and cultured by whole bone marrow adherence and density gradient centrifugation.The second generation BMSCs were inducted by 0(control group),50,100 and 200 μmol/L puerarin in vitro.The morphology of the cultured cells was observed,and the results showed that BMSCs were uniformly adherent to the wall from the suspension state of 0 hour to 24 hours;After 3days,the cell was round and blunt;After 7 days,the cells presented irregular state such as long spindle.BMSCs proliferation was accelerated after puerarin induction for 7days.After puerarin induction for 7 days,cell proliferation was accelerated and cell morphology was longer.After induction for 28 days,the cells showed a tight directional arrangement.There was no difference in BMSCs morphology induced by puerarin at different concentrations.Flow cytometry was used to identify the surface antigen of the 4th generation BMSCs.The results showed that the positive expression rates of CD29,CD45 and CD90 were 95.1%,7.4% and 93.4%,respectively.The results showed that the cells were purified BMSCs.The expression of gap connexin 43(Cx43),cardiac troponin I(c Tn I)and cardiac troponin T(c Tn T)in each group was detected by immunocytochemical assay after 4w induction.The results showed that the positive expressions of Cx43,c Tn I and c Tn T were found in all the induction groups,while the expression of Cx43,c Tn I and c Tn T were negative in the blank control group.The positive expression of the 100 μmol/L induction group was the highest compared with other groups,and the difference was statistically significant(P < 0.05).The expressions of gap connexin 43(Cx43),cardiac troponin I(c Tn I)and cardiac troponin T(c Tn T)in the control group and the 100 μmol/L induction group were detected by using laser confocal fluorescence technology after induction of 4weeks.The results showed that the expression in the 100 μmol/L induction group was positive,while the expression in the control group was negative,showing a significant difference.The expression of gap connexin 43(Cx43),cardiac troponin I(c Tn I)and cardiac troponin T(c Tn T)in each group was detected by Western blotting.The results showed that Cx43,c Tn I and c Tn T were expressed at the protein level in each group,with the highest expression in the 100 μmol/L induction group and the lowest in the control group,with statistically significant differences(P < 0.05).Real-time fluorescent quantitative PCR(RT-q PCR)technique was used to detect the expression of early myocardial transcription factor GATA-4 and myocardial cell enhancer factor 2c(MEF2c)in 1weeks,2weeks and 4weeks cells in each induction group,and the expression of T-box5 and amphiphysin-2(Bin1)in 1weeks,2weeks and 4weeks cells in the blank control group and the 100 μmol/L induction group.The results showed that the expression of each gene increased after puerarin induction compared with the control group.Although the highest expression levels of each gene appeared at different time periods,they all appeared in the 100 μmol/L induction group.Compared with each induction group,the highest expression of GATA-4 was found when the 100 μmol/L induction group induced 4weeks,and the highest expression of MEF2 c was found when the 100 μmol/L induction group induced 2weeks.Compared with the blank control group,the highest expression of T-box5 in the 100 μmol/L induction group was at 1weeks induction in the 100 μmol/L induction group,and the highest expression of Bin1 was at 4weeks induction in the 100 μmol/L induction group.Compared with other induction groups,the expression of 100 μmol/L group was the highest(P < 0.05).CONCLUSION:1.The SD rat limb bone marrow cells isolated and cultured by whole bone marrow adherence method can be purified into bone marrow mesenchymal stem cells.2.Different concentrations of puerarin can induce differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro.3.100μmol/L is the optimal concentration for puerarin to induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocytelike cells in vitro. |
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