黄精对衰老大鼠内皮祖细胞DNA损伤检测点ATM/ATR通路的影响
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摘要
目的观察黄精对衰老大鼠内皮祖细胞(endothelial progenitor cells,EPCs)体外传代培养过程中细胞周期及DNA损伤检测点毛细血管共济失调突变基因(Ataxia-telangiectasia mutated gene,ATM)/ATM与Rad3相关蛋白激酶(ATM and Rad3-related kinase,ATR)通路的影响。方法分离培养衰老大鼠骨髓EPCs并鉴定,将培养至第2代的EPCs分为4组,分别用黄精含药血清和空白对照血清继续培养至第4、6、8代,流式细胞仪检测细胞周期,RT-PCR和Western-Blot法检测细胞ATM、ATR、检测点激酶1(check point 1,Chk1)、检测点激酶2(check point 2,Chk2)mRNA及其蛋白的表达。结果 EPCs在体外传代培养过程中其细胞周期在G_1期增多,在S期减少,细胞表达ATM、ATR、Chk1、Chk2 mRNA及蛋白水平显著升高(P <0.05),经黄精干预后,EPCs在传代培养中其细胞周期在G_1期减少,在S期增多,细胞表达ATM、ATR、Chk1、Chk2 mRNA及蛋白显著降低(P <0.05)。结论黄精可通过抑制DNA损伤检测点ATM/ATR通路的活化来调节EPCs的细胞周期,干预EPCs的老化进程。
Objective To investigate the effect of Polygonati rhizoma(RP)on cell cycle and DNA damage check point Ataxia-telangiectasia mutated gene(ATM)/ATM and Rad3-related kinase(ATR)pathway in endothelial progenitor cells(EPCs)of aged rats. Methods EPCs from bone marrow of aging rats were isolated and cultured in vitro and identified. The 2 nd generation EPCs were divided into 4 groups,which were subcultured to the 4 th,6 th and 8 th passage with or without drug-serum containing RP. The cell cycle was detected by flow cytometry, the expression of ATM,ATR,check point 1(Chk1),check point 2(Chk2)mRNAs or proteins were detected by RTPCR or Western Blot. Results In the subcultures of EPCs,number of cells with cell cycle in G_1 phase increased but of those in S phase decreased, the expression of ATM, ATR, Chk1, Chk2 mRNAs or proteins increased significantly(P < 0.05). RP could decrease cell number with the cell cycle in G_1 phase and increase it in S phase,as well as decreasing the expression of ATM, ATR, Chk1, Chk2 mRNAs or proteins significantly(P < 0.05).Conclusion RP may regulate the cell cycle and delay the aging process of EPCs by inhibiting the stimulation of DAN damage check point ATM/ATR pathway.
Objective To investigate the effect of Polygonati rhizoma(RP)on cell cycle and DNA damage check point Ataxia-telangiectasia mutated gene(ATM)/ATM and Rad3-related kinase(ATR)pathway in endothelial progenitor cells(EPCs)of aged rats. Methods EPCs from bone marrow of aging rats were isolated and cultured in vitro and identified. The 2 nd generation EPCs were divided into 4 groups,which were subcultured to the 4 th,6 th and 8 th passage with or without drug-serum containing RP. The cell cycle was detected by flow cytometry, the expression of ATM,ATR,check point 1(Chk1),check point 2(Chk2)mRNAs or proteins were detected by RTPCR or Western Blot. Results In the subcultures of EPCs,number of cells with cell cycle in G_1 phase increased but of those in S phase decreased, the expression of ATM, ATR, Chk1, Chk2 mRNAs or proteins increased significantly(P < 0.05). RP could decrease cell number with the cell cycle in G_1 phase and increase it in S phase,as well as decreasing the expression of ATM, ATR, Chk1, Chk2 mRNAs or proteins significantly(P < 0.05).Conclusion RP may regulate the cell cycle and delay the aging process of EPCs by inhibiting the stimulation of DAN damage check point ATM/ATR pathway.
引文
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[2]SCHMITT E,PAQUET C,BEAUCHEMIN M,et al.DNA-damage response network at the crossroads of cell-cycle check-points,cellular senescence and apoptosis[J].J Zhejiang Univ Sci B,2007,8(6):377-397.
[3]NAKAMURA A J,CHIANG Y J,HATHCOCK K S,et a1.Both telomeric and non-telomeric DNA damage are determinants of mammalian cellular senescence[J].Epigenetics&Chromatin,2008,1(1):6.
[4]国家药典委员会.中华人民共和国药典(一部)[S].北京:化学工业出版社,2005:215.
[5]刘小群,乔田奎,陈伟,等.ATM蛋白激酶依赖性信号转导通路研究进展[J].复旦学报(医学版),2012,39(4):433-437.
[6]程晋,邹仲敏.DNA损伤修复及细胞周期检控点激活的研究进展[J].国际放射医学核医学杂志,2009,33(6):360-364.
[7]SANCAR A,LINDSEY-BOLTZ L A,UNSAL-KACMAZ K,et al.Molecular mechanisms of mammalian DNA repair and the DNAdamage checkpoints[J].Annu Rev Biochem,2004,73:39-85.
[8]STRAEKER T H,USUI T,PETRINI J H.Taking the time to make important decisions:the checkpoint effector kinases Chkl and Chk2and the DNA damage response[J].DNA Repair(Amst),2009,8(9):1047-1054.
[9]安红梅,史云峰,史秀峰,等.补肾填精方对D-半乳糖衰老模型大鼠脑细胞周期相关蛋白表达的影响[J].中南药学,2009,7(3):161-164.
[10]陈川,迟惠英,郁志华,等.首参颗粒对动脉粥样硬化大鼠血管细胞与外周血白细胞端粒和端粒酶的影响[J].中西医结合学报,2012,10(6):667-673.
[11]卓勤,徐琦.补肾益精药物对老年小鼠DNA双链结构及损伤修复能力影响[J].中国中医基础医学杂志,1998,4(9):40-44.