| Background:With the development of society and improvement of living standard, cardiovascular disease(CVD) has become the leading cause of death. Cardiomyocyte injuries caused by many reasons lead to the irreversible loss of cardiomyocyte, and finally cause heart failure. Although cardiac stem cells exist in adult heart, it is insufficient to compensate cell death by heart disease. Interventional medicine improves the quality of life, however, current medical strategies cannot cross the hurdle of cardiac repair. Regeneration medicine brings the new hope, especially induced pluripotent stem cells(i PSCs) by reprogramming adult cells, which have multiple differentiation potential. Cardiac differentiation of i PSCs may provide a reliable cell source for cardiac repair. So research and application of i PSCs are rapidly focused by scientists. Now researchers have successfully induced mi PSCs to differentiate into cardiomyocyte, but the low differentiation efficiency exsits as a great obstacle which significantly hampers the clinical application of i PSCs. Many studies have shown that growth differentiation factor 11(GDF-11) can activate TGF-? signaling pathway which have a close relationship with cardiac differentiation of i PSCs. But research about the positive effect of GDF-11 on cardiac differentiation of i PSCs has not been reported as yet.Objective:To investigate the effect of GDF-11 on cardiac differentiation of mi PSCs and the possible molecular mechanisms in order to provide experimental bases for clinical treatment of i PSCs.Method:1. Preparation of mouse embryonic fibroblast as the feeder cell of mouse-induced pluripotent stem cells(mi PSCs). 2. Cultivation of mi PSCs. 3. The formation of embryonic bodies(EBs) and cardiac differentiation of mi PSCs. 4. Optimization of GDF-11 concentration. 5. Detect the stage-specific gene expression of RNA level of cardiac differentiation by real-time PCR and analyze the variance. 6. Observe the expression level of c Tn I by Western blot. 7. Examine the expression level of c Tn I by immunoflurorescence staining. 8. Detect the expression of proteins related to TGF-? signaling pathway and PGC-1? as a mitochondrial functional protein.Result: 1. mi PSCs on the following day after recovery exhibited stereoscopic round cell clusters and lined up tightly in the clusters. EBs formed from mi PSCs with hanging drop method showed the similar size. Contracting mi PSCs derived cardiomyocytes were observed until day 8 of differentiation. 2. 10ng/ml was proved to be the most efficient concentration through comparing the expression of c Tn I and ?-MHC.3. The expression of cardiac specific markers was detected by real-time PCR, which show that the expressions of Oct-4 in GDF-11 group is significantly lower than control group(P<0.01), GDF-11 enhanced the expression of flk-1 and Nkx2.5(P<0.01) and the expression of c Tn T was significantly improved in GDF-11 group(P<0.01).4. Beating areas were found in both groups, but GDF-11 group had a higher percentage of beating EBs(P<0.01).5. The expression of c Tn I, as a kind of myocardial structural protein, analyzed by Western blot, was promoted by GDF-11. Immunoflourensence staining showed more c Tn I positive cells in GDF-11 group(P<0.01).6. Compared with control group, the expression of TGF-?, smad2 and PGC-1? had a robust improvement in GDF-11 group(P<0.05).Conclusion: GDF-11 significantly enhanced the expression of early stage cardiac transcription factor Nkx2.5 and cardiac specific marker c Tn I and facilitated the process of cardiac differentiation from mi PSCs, and further improved the differentiation efficiency. The effect of GDF-11 might have a direct correlation with TGF-? signaling pathway and mitochondrial function. |
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