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

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

背景:5-氮杂胞苷是诱导骨髓间充质干细胞分化为心肌样细胞的有效化学试剂。目的:观察 P53 特异性抑制剂 PFT-α阻断 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。方法:分离 SD 大鼠骨髓间充质干细胞,取第 3 代细胞分为 4 组,即正常对照组、5-氮杂胞苷组、PFT-α组、PFT-α+5-氮杂胞苷组。观察骨髓间充质干细胞培养传代过程中细胞形态变化,应用流式细胞仪鉴定骨髓间充质干细胞表面抗原,MTT 法测定诱导后各组骨髓间充质干细胞增殖能力,以流式细胞仪检测诱导后各组细胞凋亡率,采用 Western blot 法测定诱导后P53、P21 蛋白表达情况。结果与结论:原代培养的骨髓间充质干细胞 2 周形成集落,传代细胞体积变大,呈长梭形,排列趋一致。骨髓间充质干细胞表面抗原 CD44,CD45 阳性表达率分别为(89.98±1.29)%,(2.14±0.22)%。MTT 结果显示,当 PFT-α浓度≤20 μmol/L 时,对骨髓间充质干细胞的增殖有一定促进作用;而当其浓度达到 40 μmol/L 时,PFT-α则可明显抑制骨髓间充质干细胞的增殖。培养第 5,7 天时,与其他 3 组比较,5-氮杂胞苷组对骨髓间充质干细胞增殖表现出明显抑制(P<0.01)。流式细胞仪结果显示,5-氮杂胞苷组骨髓间充质干细胞凋亡率显著高于其他 3 组(P<0.01)。Western blot 结果显示,各组均有 P53、P21 蛋白的表达,以 5-氮杂胞苷组表达量明显增多。提示通过 P53 特异性抑制剂 PFT-α阻断 P53-P21 蛋白通路,能显著减少 5-氮杂胞苷诱导的骨髓间充质干细胞的凋亡,促进 5-氮杂胞苷诱导的骨髓间充质干细胞增殖。

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（P0.01）;in 7 days,it also showed such differences（P0.01）,and BMSCs proliferation of PFT-α group and 5-AZA ＋ PFT-α group was significantly better than blank control group（P0.01）.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（P0.01）.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