Experimental Study Of Differentiating Bone Marrow Mesenchymal Stem Cells Into Cardiomyocyte-like Cells By P53 Inhibitor(PFT-α)

BackgroundIn recent years, the incidence of Coronary Heart Disease increases year by year, particularly ischemic cardiomyopathy caused by myocardial infarction which has become the major effect to human health. Recent studies have shown that a small amount of cardiomyocytes proliferate after myocardial infarction, but the cells of proliferation were too few to make up the apoptosis of a large number cardiomyocytes at myocardial infarction. Therefore, a large number of cardiomyocytes necrosis caused by myocardial infarction eventually lead to the occurrence of ischemic cardiomyopathy, leading to serious heart failure and a variety of cardiovascular complications, the five-year mortality rate reaches up to 45%. Despite of the increasing improvement of cardiovascular drug therapy, coronary artery bypass graft and cardiac interventional technology, the mortality of cardiovascular disease remains high. So how to repair necrosis cardiomyocytes after myocardial infarction are always serious challenges faced by researchers and clinicians. Stem cell transplantation has been suggested as a promising approach for the treatment of cardiovascular disease. Currently, bone marrow mesenchymal stem cells(BMMSCs) is the most popular among adult stem cells. But how to obtain a sufficient number of cardiomyocyte-like cells becomes a hot spot and the difficulty of research. With further research, a series of problems appeared in the process of BMMSCs transplantation treating the ischemic cardiomyopathy after myocardial infarction will be solved.Objectives1. To explore separation, purification, passage and identification of the rat BMMSCs in vitro.2. To observe the effect of PFT-αon the proliferation and apoptosis of the rat BMMSCs and its possible mechanism.3. To investigate the effects of PFT-αon differentiation rate of BMMSCs differentiation into cardiomyocyte-like cells in vitro and its possible mechanism.Methods1. Isolation, culture, purification and identification of the rat BMMSCs in vitro: Rats were killed by cervical dislocation and long bone of lower limbs were taken. Other cells mixed in BMMSCs were removed by density gradient centrifugation combined differential adhesion method, then the purified BMMSCs were obtained. The morphological changes were observed under phase contrast microscope during the culture and passage of BMMSCs.The positive surface marker CD44 and negative marker CD45 of cells were detected for the identification of BMMSCs with flow cytometry.2. The effects of PFT-αon the proliferation and apoptosis of the rat BMMSCs: The fourth passaged BMMSCs were used. The cytotoxicity of PFT-αwas detected by MTT assay. Then the cells were divided into 4 groups:①Control group (CON): cells were untreated;②5-AZA group: cells were treated by 5-AZA at 10μmol/L for 24 h;③PFT-αgroup: cells were treated by PFT-αdissolved in DMSO at 20μmol/L for 24 h;④PFT-α+5-AZA group: cells were treated by PFT-αat 20μmol/L and 5-AZA at 10μmol/L for 24 h. The cell proliferation ability was observed with MTT assay. The effect of PFT-αon apoptosis of BMMSCs was examined by flow cytometer. The expression level of P53 and P21 protein were examined by western blotting.3. PFT-αinduced BMMSCs to differentiate into cardiomyocyte-like cells: The fourth passaged BMMSCs were divided into 4 groups:①Control group (CON): cells were untreated;②5-AZA group: cells were treated by 5-AZA at 10μmol/L for 24 h;③PFT-αgroup: cells were treated by PFT-αdissolved in DMSO at 20μmol/L for 24 h;④PFT-α+5-AZA group: cells were treated by PFT-αat 20μmol/L and 5-AZA at 10μmol/L for 24 h. Conventional medium was replaced 24 h after induction and continue to culture for 4 w. The morphological changes of different time points were observed under phase contrast microscope. The expression of cardiomyocyte specific proteins cTnI and CX-43 were detected by immunofluorescence staining. The level of P53 and P21 protein was examined by western blotting. The induction ratio of cardiomyocyte-like cells was examined by flow cytometer.Results1. Morphological characteristics and identification of the rat BMMSCs in vitro: Primary BMMSCs were smaller and had varied shapes, most of cells were oval or short spindle. After 7 d, the adherent cells rapidly proliferated and formed large or small colonies. After passage, the cells were larger than the original cells and showed elongated spindle. To provide quantitative evaluation of these cells we performed flow cytometry analysis of BMMSCs of the fourth passage and detected that (89.98±1.29)% of cells expressed surface marker CD44 but only (2.14±0.22)% of cells expressed surface marker CD45, confirming that we obtained purified BMMSCs.2. The effects of PFT-αon the proliferation and apoptosis of the rat BMMSCs: MTT assay showed that PFT-αpromoted the proliferation of BMMSCs in a dose-dependent manner in the range of 0-20μmol/L, although the effects on the proliferation were not statistically significant. However, PFT-αsignificantly inhibited the proliferation of BMMSCs at the concentration of 40 and 80μmol/L. These results demonstrate that low dose of PFT-αpromoted the proliferation of BMMSCs but high dose of PFT-αhad significant cytotoxic effects. MTT showed that in the first three days the proliferation of every groups had no significant difference. At the five day and seven day, the proliferation in 5-AZA group was the lower compared to other groups (P<0.01). And the proliferation in PFT-αgroup and 5-AZA+PFT-αgroup had statistical difference compared with CON group (P<0.01). Flow cytometry analysis was used to test the apoptosis rate. The results showed that the apoptosis rate in 5-AZA group was highest compared to other groups (P<0.01). However, the apoptosis rate in CON group, PFT-αgroup and 5-AZA+PFT-αgroup had no statistical difference (P﹥0.05). Western blotting analysis showed that P53 and P21 expression of 5-AZA group was the highest with significant difference compared to other groups (P<0.001). However, P53 and P21 expression of PFT-αgroup was significantly reduced with significant difference compared to the CON group and the 5-AZA+PFT-αgroup(P<0.01), there was no significant difference between CON group and 5-AZA+PFT-αgroups (P﹥0.05). 3. PFT-αinduced BMMSCs to differentiate into cardiomyocyte-like cells: The morphological changes of BMMSCs after induction in every group. Except for CON group, cell volume and nuclear increased and most of the cells became spindle length after induction by 2 w. At 4 w after induction, BMMSCs represented as closely packed elongated cells with uniform morphology and had myotube-like structure. Immunofluorescence staining showed that at 1 w after induction, no expression of cTn I and CX-43 was observed in CON group, but a little expression of cTn I and CX-43 were observed in other three treatment groups. 4 w after induction, CON group olny had little expression of cTn I and CX-43, while the other three groups demonstrated strong expression of cTn I and CX-43 and the cells developed myotube-like structure. Western blotting analysis showed that at 1 w after induction, cTn I expression in 5-AZA group, PFT-αgroup and 5-AZA+PFT-αgroup was low, but no cTn I expression was observed in CON group. At 4 w, only a little expression of cTn I was observed in CON group, but in three treatment groups cTn I expression were significantly increased and much higher than CON group. cTn I expression in every group at 4 w was higher than that of at 1 w. Flow cytometry determined the differentiation rate of cardiomyocyte-like cells and the results showed that differentiation rate of CON group was (2.34±0.91)%, 5-AZA group was (21.47±5.41)%, PFT-αgroup was (29.85±1.96)%, 5-AZA+PFT-αgroup was (30.97±5.74)%. 5-AZA group, PFT-αgroup and 5-AZA+PFT-αgroup was significant higher than CON group (P<0.01). In addition, the differentiation rate in PFT-αgroup and 5-AZA+PFT-αgroup was significantly higher than 5-AZA group (P<0.01), but there was no significant difference between PFT-αgroup and 5-AZA+PFT-αgroup (P﹥0.05). Western blotting analysis showed that at 1 w the expression of P53 and P21 in 5-AZA group were strongest compare to other groups (P<0.01), but no expression was observed in PFT-αgroup. There was no statistical difference between PFT-αgroup and 5-AZA+PFT-αgroup (P﹥0.05). At 4 w, the expression of P53 and P21 in 5-AZA group were still strong, and the expression in other three groups were much higher than that of at 1 w. 5-AZA group, PFT-αgroup and 5-AZA+PFT-αgroup was significant higher than CON group (P<0.05). 5-AZA group and PFT-αgroup had significant difference (P<0.01). The expression of P53 and P21 in PFT-αgroup and 5-AZA+PFT-αgroup at 4w were much higher than that of at 1 w (P<0.05). These results indicate that modulation of P53 and P21 expression by PFT-αmay contribute to the high rate of differentiation of BMMSCs induced by PFT-α(P﹥0.05).Conclusion1. The purified BMMSCs were obtained by density gradient centrifugation combined differential adhesion method. The purified BMMSCs could be identified by detecting positive surface antigen CD44 and negative surface antigen CD45.2. Low dose of PFT-αpromoted the proliferation of BMMSCs but high dose of PFT-αhad significant cytotoxic effects. PFT-αcould inhibit the apoptosis of BMMSCs.3. PFT-αcould induce BMMSCs to differentiate into cardiomyocyte-like cells, and differentiation rate increased by 39% than 5-AZA group (P﹤0.01).4. PFT-αplays a role through inhibition of P53-P21 protein pathway.