| Objective: To investigate the effect of Elabela on the directional differentiation of human umbilical cord Wharton's Jelly mesenchymal stem cells mesenchymal stem cells(WJMSCs)into cardiomyocyte-like cells and the expression of APJ.Methods: Resuscitated the frozen human umbilical cord-derived WJ-MSCs,the cells were subcultured when the confluence of cells reached above 90%.The passaged cells were cultured on a cell well plate.When the confluence reached 80-90%,a certain concentration of 5-azacytidine(5-aza)medium was added to induce the cells for 24 hours,and then divided the cells into two groups: the experimental group and the control group.The experimental group medium was replaced with 10% fetal bovine serum mixed with DMEM low-glucose medium containing Elabela to continue culturing the cells,the control group medium was replaced with 10% fetal bovine serum mixed with DMEM low-glucose medium to continue culturing the cells.When cultured to 28 days,the expression of Nkx2.5,a specific transcription factor of cardiomyocytes,was detected by cellular immunofluorescence staining.In addition,the morphological changes and growth of the cells were observed and recorded at the 7d,14 d,21d and 28 d of the induction.Besides,collected two groups' the total RNA and total protein of cells at each stage,detected by quantitative real-time PCR and Western blot techniques for the expression of myocardial specific markers Connexin 43,c Tn T,Nkx2.5 m RNA and protein.Collecting the cells of the experimental group and the control group,then measuring the expression of APJ at the 7d,14 d,21d and 28 d by using quantitative real-time PCR technology.In addition,the expression of the target gene APJ on the 28 days of differentiation was detected by flow cytometry.Results: During the process of differentiation of human umbilical cord WJ-MSCs directed cardiomyocyte-like cells,the number of cardiomyocyte-like cells in the experimental group and the control group under the inverted microscope gradually increased during the differentiation process.The expression levels of specific marker proteins c Tn T and Nkx2.5 were significantly higher than those of the control group,and there was a significant difference(P <0.05).On the 21 st and 28 th days,the expression of c Tn T m RNA in the experimental group was increased to 124% and 113%,respectively,and the expression of Nkx2.5 m RNA was increased to 224% and 250%;After reaching the highest level,there were different degrees of decline after the 21 st day.There was no significant difference in the expression levels of Connexin43 m RNA and protein in the experimental group and the control group at each stage(P> 0.05).The cellular immunofluorescence technique of the experimental group and the control group detected positive expression of myocardium-specific transcription factor Nkx2.5.The results of real-time fluorescence quantitative PCR showed that the two groups of cells had the target gene APJ m RNA expression at each time node of differentiation,and there was no significant difference in the expression of APJ between the two groups on the 7th day(P> 0.05).On the 14 th,21d,and 28 d,the APJ expression on the experimental group of the amount was significantly higher than that of the control group(P <0.05),especially the 14 th day,the difference was the most significant,up to 647%.The results of flow cytometry showed that the experimental group and the control group induced APJ expression on the 28 th day of differentiation.Compared with the undifferentiated human umbilical cord WJ-MSCs,the expression of APJ increased by 33.03% and 61.65% respectively,increased by 186.7%,with significant statistical difference(P <0.05).Conclusion: Human umbilical cord WJ-MSCs were successfully differentiated into cardiomyocytes under the induction of 5-aza.Elabela promoted the differentiation of human umbilical cord WJ-MSCs into myocardial-like cells.Elabela promoted APJ expression during the differentiation of human umbilical cord WJ-MSCs into myocardiallike cells. |
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