Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (14): 2482-2486.doi: 10.3969/j.issn.1673-8225.2011.14.003

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P53-P21 protein pathway impacts on proliferation and apoptosis of 5-azacytidine induced bone marrow mesenchymal stem cells

Yan Xue-bo, Lü An-lin , Liu Bo-wu, Hou Jing, Huang Wei, Li Yao   

  1. Department of Cardiology, Xijing Hospital of Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
  • Received:2010-10-12 Revised:2010-12-14 Online:2011-04-02 Published:2013-11-02
  • Contact: Lü An-lin, Doctor, Associate professor, Department of Cardiology, Xijing Hospital of Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China lvanlin@fmmu.edu.cn
  • About author:Yan Xue-bo★, Studying for master’s degree, Attending physician, Department of Cardiology, Xijing Hospital of Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China

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Abstract:

BACKGROUND: 5-azacytidine (5-AZA) is an effective reagent to induce bone marrow mesenchymal stem cells ( BMSCs) differentiating to cardiomyocytes.
OBJECTIVE: To investigate PFT-α blocked P53-P21 protein pathway impacts on proliferation and apoptosis of 5-AZA induced BMSCs.
METHODS: BMSCs were isolated from bone marrow of SD rats by density gradient centrifugation. The third passage cells were divided into 4 groups: (1) control group (CON); (2) 5-AZA group; (3) PFT-α group; (4) PFT-α + 5-AZA group. Morphological changes of BMSCs were observe, surface antigen of BMSCs with flow cytometry was identified, proliferation capacity of BMSCs in each group was determined by MTT, apoptosis rate of BMSCs in each group after induction was detected with flow cytometry, and P53, P21 protein expression of BMSCs in each group after induction was determined with the Western blotting method.
RESULTS AND CONCLUSION: BMSCs of primary culture form colonies at 2 weeks, passaged cells became larger, elongated spindle, arranged the same trend. The results of Flow cytometry showed CD44 expression of BMSCs was (89.98±1.29)%, CD45 positive expression rate was (2.14±0.22)%. MTT results showed that when the concentration of PFT-α was equal to or less than  20 μmol/L, promoted proliferation of BMSCs, and when concentration reached 40 μmol/L, significantly inhibited proliferation of BMSCs; in 5 days, 5-AZA group significantly inhibited proliferation of BMSCs. Compared with the control group, PFT-α group and 5-AZA + PFT-α group, there was a significant difference (P < 0.01); in 7 days, it also showed such differences (P < 0.01), and BMSCs proliferation of PFT-α group and 5-AZA + PFT-α group was significantly better than blank control group (P < 0.01). The results of Flow cytometry showed, BMSCs apoptosis rate of 5-AZA group was the highest as compared with that of the control group, PFT-α group and 5-AZA + PFT-α group (P < 0.01). Western blotting results showed there was P53, P21 protein expression in each group, the expression of 5-AZA group was significantly increased, P53, P21 protein expression of PFT-α group and 5-AZA+PFT-α group was significantly reduced. By P53 inhibitor PFT-α blocking, P53-P21 protein pathway can significantly reduce the apoptosis of 5-AZA-induced BMSCs, and promote proliferation of 5-AZA-induced BMSCs.

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