Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-likecells induced by P53 specific inhibitor p-fifty three inhibitor-alpha

doi:10.3969/j.issn.2095-4344.2013.01.002

Abstract

Abstract:

BACKGROUND: Latest researches have shown that P53 can significantly increase the differentiation rate of stem cells by locking the protein pathway P53-P21. While 5-azacytidine can inhibit cell proliferation and lead to apoptosis by activating the P53-P21 protein pathway.
OBJECTIVE: To investigate the effects of P53 specific inhibitor p-fifty three inhibitor-α on differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells.
METHODS: Bone marrow mesenchymal stem cells were separated from Sprague-Dawley rats for culturing and passage. Passage 4 bone marrow mesenchymal stem cells were divided into four groups: normal control group, p-fifty three inhibitor-α group, 5-azacytidine and p-fifty three inhibitor-α+5-azacytidine group.
RESULTS AND CONCLUSION: Primary bone marrow mesenchymal stem cells were smaller, and after passaged and induced, the cells were larger than primary cells and exhibited spindle-shaped appearance and arranged in order. MTT assay showed that p-fifty three inhibitor-α at 0-20 μmol/L could reduce the apoptosis of bone marrow mesenchymal stem cells. Identification results of cardiomyocyte-like cells showed that after induced for 4 weeks, little expression of cardiac troponin Ⅰ and CX-43 could be seen in the normal control group, and the expression of cardiac troponin Ⅰ and CX-43 in the other three groups was strong. After induced for 4 weeks, the differentiation rate of cardiomyocyte-like cells in the p-fifty three inhibitor-α and p-fifty three inhibitor-α+ 5-azacytidine group was significantly higher than that in the 5-azacytidine group. Western blotting analysis showed that at 1 week after induction, the expression of P53 and P21 in 5-azacytidine group was strongest compared with the other groups, but no expression was observed in p-fifty three inhibitor-α group. At 4 weeks after induction, the expression levels of P53 and P21 in 5-azacytidine group, p-fifty three inhibitor-α group and p-fifty three inhibitor-α+5-azacytidine group were higher than those in the normal control group. The expression levels of P53 and P21 in p-fifty three inhibitor-α group and p-fifty three inhibitor-α+5-azacytidine group at 4 weeks after induction were higher than those at 1 week after induction. It indicates that P53 specific inhibitor p-fifty three inhibitor-α can significantly induce the apoptosis, promote the proliferation of bone marrow mesenchymal stem cells through blocking P53-P21 protein pathway, and inhibit bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells.

Key words: stem cells, bone marrow stem cells, p-fifty three inhibitor-α, bone marrow mesenchymal stem cells, P53-P21 protein pathway, 5-azacytidine, cardiomyocyte-like cells, stem cell photographs-containing paper

CLC Number:

R394.2

Yan Xue-bo, Hu Zhao-hui, Du Yun-hua, Lü An-lin, Xing Yu-jie. Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-likecells induced by P53 specific inhibitor p-fifty three inhibitor-alpha[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(1): 9-16.

Figures/Tables 7

References

[1] Elizabeth GN. Cardiovascular disease. New England J Med. 2003;349:60-72.

[2] Cao F, Sun DD, Li CX, et al. Long-term myocardial functional improvement after autologous bone marrow mononuclear cells transplantation in patients with ST-segment elevation myocardial infarction: 4 years follow-up. Eur Heart J. 2009; 30(16):1986-1994.

[3] Deng W, Obrocka M, Fischer I, et al. In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem Biophys Res Commun. 2001;282(1): 148-152.

[4] Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 1995;18:1417-1426.

[5] Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest. 1999;103(5):697-705.

[6] Hong H, Takahashi K, Ichisaka T, et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature. 2009;460: 1132-1136.

[7] Armesilla DA, Elvira G, Silva A. p53 regulates the proliferation, differentiation and spontaneous transformation of mesenchymal stem cells. Exp Cell Res. 2009;315: 3598-3610.

[8] Pulukuri SM, Rao JS. Activation of p53/p21Waf1/Cip1 pathway by 5-aza-2'-deoxycytidine inhibits cell proliferation, induces pro-apoptotic genes and mitogen-activated protein kinases in human prostate cancer cells. Int J Oncol. 2005;26(4): 863-871.

[9] Zhu WG, Hileman T, Ke Y, et al. 5-Aza-2,-deoxycytidine Activates the p53/p21Waf1/Cip1 Pathway to Inhibit Cell Proliferation. J Biol Chem. 2004;279(15): 15161-15166.

[10] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.

[11] Bergmann O, Bhardwaj RD, Bernard S, et al. Evidence for Cardiomyocyte Renewal in Humans. Science. 2009; 324 (5923): 98 -102.

[12] Li X,Yu X, Lin Q, et al. Bone marrow mesenchymal stem cells differentiate into functional cardiac phenotypes by cardiac microenvironment. J Mol Cell Cardiol. 2007;42(2):295-303.

[13] Wang JS, Shunr Tim D, Galipeeall J, et al. Marrou Stramol cells for cellular Cardiomyoplasty :feasibillty and potential dinical advantages. Thorae Cardiovasc Surg. 2000;120(62): 999-1005.

[14] Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation. 2002;106 (15):1913-1918.

[15] Janssens S, Dubois C, Bogaert J, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction:double-blind, randomised controlled trial. Lancet. 2006;367(9505): 113-121.

[16] Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone marrow cell transfer after myocardial infarction: The BOOST randomised controlled clinical trial. Lancet. 2004; 364(9429) :141-148.

[17] Lago N, Trainini J, Christen A, et al. Mononuclear bone marrow stem cells implant as an alternative treatment in non-ischemic dilated cardiomyopathy. J Am Coll Cardiol. 2006;47(4 Suppl. A):1A-384A.

[18] Wang J, Xie X, He H, et al. A prospective, randomized, controlled trial of autologous mesenchymal stem cells transplantation for dilated cardiomyopathy. Chin J Cardiol. 2006;34(2):107-111.　

[19] Komarova EA, Gudkov AV. Suppression of p53: a new app roach to overcome side effects of antitumor therapy. Biochemistry (Mosc).2000;65:41-48.

[20] Schneider SR, Diab AM, Rohrbeck A, et al. 5-aza-Cytidine is a potent inhibitor of DNA Methyltransferase 3a and induces apoptosis in HCT-116 colon cancer cells via Gadd45- and p53-Dependent Mechanisms. J Pharmacol Exp Ther. 2005; 312(2):525-536.

[21] Yan XB, Lü AL, Liu BW, et al.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(14):2482-2486.燕学波,吕安林,刘博武,等. P53-P21蛋白通路对5-氮杂胞苷诱导的大鼠骨髓间充质干细胞增殖和凋亡的影响[J].中国组织工程研究与临床康复,2011,15(14):2482-2486.

[22] Levine AJ, Finlay CA, Hinds PW. P53 is a Tumor Suppressor Gene. Cell. 2004;116:67-69.

[23] Schuler M, Green DR. Transcription, apoptosis and P53: catch-22. Trends Genet. 2005;21(3): 182-187.

[24] Xuwan L, Chu CC, Jinping G, et al. Pifithrin-α protects against doxorubicin-induced apoptosis and acute cardiotoxicity in mice. Am J Physiol Heart Circ Physiol. 2004;286: 933-939.

[25] Shave R, Baggish A, George K, et al. Exercise-induced cardiac troponin elevation: evidence, mechanisms, and implications. J Am Coll Cardiol. 2010;56(3):169-176.

[26] Janczarska K, Kie?-Wilk B, Leszczyńska-Go?abek I, et al. The role of connexin 43 in preconditioning. Impact on mitochondrial function. Kardiol Pol. 2010;68(1):91-96.