摘要
目的探讨核转录因子阴阳1(yin yang 1,YY1)对体外缺血缺氧诱导的心肌细胞凋亡的抑制作用及其机制。方法体外分离培养新生大鼠心肌细胞,利用无糖培养基及含95%N2+5%CO2混合气体的缺氧装置(Billups-rothenberg)诱导建立缺血缺氧心肌细胞凋亡模型,通过建立过表达YY1重组质粒并将其转染入心肌细胞内,观察YY1对缺血缺氧诱导的心肌细胞凋亡的影响。实验分为对照组、YY1组、缺血缺氧组、缺血缺氧+YY1组、缺血缺氧+LY294002组、缺血缺氧+YY1+LY294002等6组(n=4)。CCK-8法检测各组心肌细胞活力,TUNEL法检测各组心肌细胞凋亡情况,f QT-PCR检测YY1 mRNA表达水平,Western blot法检测YY1、磷酸化丝氨酸苏氨酸激酶(p-Aktser473)及生存素(Survivin)蛋白表达水平。结果与对照组相比,缺血缺氧组在缺血缺氧刺激6 h后心肌细胞活力下降、凋亡率明显增高[(0.55±0.02)vs(1.14±0.02)、(38.25±1.65)%vs(1.38±0.15)%,P<0.01]。缺血缺氧前预转染YY1重组质粒后,与缺血缺氧组相比,缺血缺氧+YY1组心肌细胞活力上升、凋亡率降低、p-Aktser473和Survivin蛋白表达增加[(0.78±0.03)vs(0.55±0.02)、(14.50±1.56)%vs(38.25±1.65)%、p-Aktser473相对灰度值:(0.82±0.03)vs(0.28±0.03)、Survivin相对灰度值:(0.93±0.02)vs(0.36±0.02),P<0.01]。经PI3K/Akt信号通路特异性抑制剂LY294002干预后,缺血缺氧+YY1+LY294002组与缺血缺氧+YY1组相比,心肌细胞活力下降、凋亡率增加、p-Aktser473和Survivin蛋白表达显著降低[(0.62±0.02)vs(0.78±0.03)、(27.75±1.12)%vs(14.50±1.56)%,p-Aktser473相对灰度值:(0.43±0.02)vs(0.82±0.03),Survivin相对灰度值:(0.27±0.02)vs(0.93±0.02),P<0.01]。结论核转录因子YY1可抑制缺血缺氧诱导的心肌细胞凋亡,且该作用与PI3K/Akt信号通路的激活相关。
Objective To determine the effects of a transcription factor,Yin Yang 1（ YY1） on the apoptosis of cardiomyocytes induced by ischemic hypoxia and investigate the related mechanisms. Methods Cardiomyocytes were isolated from neonatal rat heart tissue and then cultured in vitro. Sugar-free culture medium and Billups-rothenberg（ 95% N2＋ 5% CO2） was used to induce the apoptosis of cardiomyocytes.Plasmid of YY1 overexpression was established and then tansfected into the cardiomyocytes. Then the cells were divided into 6 groups of cells,that is,normal control group,YY1 group,ischemic hypoxia group,ischemic hypoxia ＋ YY1 group,ischemic hypoxia ＋ LY294002 group and ischemic hypoxia ＋ YY1 ＋ LY294002 group. The viability of cardiomyocytes of above groups was measured by CCK-8 assay. Cell apoptosis was measured with TUNEL assay. The mRNA expression of YY1 was detected by real-time quantitative PCR. The expression levels of YY1,phosphorylated Aktser473 and survivin were detected by Western blotting. Results Compared with the normal control group,the viability was significantly decreased（ 0. 55 ± 0. 02 vs 1. 14 ±0. 02,P〈0. 01） and the apoptosis increased [（ 38. 25 ± 1. 65） % vs（ 1. 38 ± 0. 15） %,P〈0. 01] in the ischemic hypoxia treatment group after 6 hours＇ ischemia-hypoxia stimulation. But transfection of YY1 overexpression plasmid prior to ischemic hypoxia stimulation could significantly reverse the phenomena [0. 78 ±0. 03 vs 0. 55 ± 0. 02,（ 14. 50 ± 1. 56） % vs（ 38. 25 ± 1. 65） %,P〈0. 01],and enhanced the protein levels of p-Aktser473 and survivin（ 0. 82 ± 0. 03 vs 0. 28 ± 0. 03,0. 93 ± 0. 02 vs 0. 36 ± 0. 02,P〈0. 01）.However,the inhibitor of PI3 K / Akt signal pathway,LY294002,suppressed the cell viability（ 0. 62 ± 0. 02 vs0. 78 ± 0. 03,P〈0. 01） and apoptosis [（ 27. 75 ± 1. 12） % vs（ 14. 50 ± 1. 56） %,P〈0. 01 ],and repressed the expression levels of p-Aktser473（ 0. 43 ± 0. 02 vs 0. 82 ± 0. 03,P〈0. 01） and survivin（ 0. 27 ±0. 02 vs 0. 93 ± 0. 02,P〈0. 01） induced by YY1 plasmid transfection. Conclusion YY1 inhibits the apoptosis in ischemic hypoxia-induced cardomyocytes,which may be mediated by activation of PI3 K / Akt signal pathway.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2016年第9期932-937,共6页
Journal of Third Military Medical University
基金
重庆市自然科学基金重点项目(CSTC2011JJB10020)~~
