摘要
目的探讨蛋氨酸亚砜还原酶B1(methionine sulfoxide reductase B1,MsrB1)基因沉默对过氧亚硝酸根(ONOO-)诱导的人晶状体上皮细胞(human lens epithelial cells,HLEC)细胞周期的影响和ERK信号通路在其中的调节作用。方法将培养的HLEC分为两组:正常组和MsrB1基因沉默组,分别用0μmol·L-1、100μmol·L-1、200μmol·L-1和300μmol·L-1 ONOO-处理20 min后,继续培养12 h。用RT-PCR法检测MsrB1基因表达水平变化,流式细胞仪检测细胞周期水平变化,Western blot法检测MsrB1基因沉默和ONOO-对pERK表达水平的影响。结果 300μmol·L-1 ONOO-处理HLEC会导致MsrB1表达水平显著下降(P<0.01),当MsrB1基因沉默细胞经300μmol·L-1ONOO-处理后,MsrB1表达水平进一步显著下调(P<0.05)。经200μmol·L-1或300μmol·L-1 ONOO-处理,HLEC细胞周期分别阻滞在G2/M期和S期,当MsrB1基因沉默细胞经200μmol·L-1或300μmol·L-1 ONOO-处理后,更多的细胞阻滞在G2/M期或S期(P<0.05)。pERK检测结果表明,MsrB1基因沉默前后经ONOO-处理导致的细胞周期阻滞和pERK表达水平显著下降有关(P<0.01)。结论 ONOO-可以下调HLEC MsrB1表达水平,MsrB1基因沉默细胞经ONOO-处理,pERK表达水平显著下降,导致更多细胞阻滞在G2/M期和S期。
Objective To investigate the effects of methionine sulfoxide reductases B1(MsrB1) gene silencing on peroxynitrite-induced cell cycle arrest and the role of ERK signaling in human lens epithelial cells(HLEC).Methods HLEC were cultured in DMEM medium and treated with MsrBl siRNA or ONOO-(0 μmol·L^-1,100μmol·L^-1 200 μmol·L^-1and 300 μmol·L^-1)for 12 hours.Then the effects of ONOO^- on the expressing levels of MsrBl were assessed by Real-time PC R and cell cycle was quantified using flow cytometry,as well as the levels of pERK were measured by Western blot.Results The levels of MsrBl mRNA in HLEC were significantly decreased after treatment with 300 μmol · L^(-1)ONOO^-(P〈0.01),when MsrB1-gene-silenced cells were exposed to 300 μmol · L^(-1)ONOO^-,the levels of MsrB1 mRNA were further decreased(P〈0.05);The results demonstrate that treatment with 200 μmol·L^-1 or 300 μmol·L^-1ONOO ^resulted in G2/M and S cell cycle arrest,respectively,when MsrB1-gene-silenced cells were exposed to 200 μmol·L^-1 or 300μmol·L^-1 ONOO^-,more HLEC occurred G2/M and S cell cycle arrest(P〈0.05).The results that cell cycle arrest in HLEC treated with ONOO^- before and after MsrB1 gene silencing was consistent with the results that the levels of pERK were significantly decreased(P〈0.01).Conclusion The levels of MsrBl mRNA in HLEC are down-regulated by ONOO^-,when MsrB1-gene-silenced cells are treated with ONOO^-,the levels of pERK are significantly decreased,and then resulted in G2/M and S cell cycle arrest in more HLEC.
出处
《眼科新进展》
CAS
北大核心
2015年第11期1021-1024,共4页
Recent Advances in Ophthalmology
基金
贵州省科学技术基金资助(编号:黔科合J-2014-2028)
贵阳医学院博士启动基金(编号:YJ2014-8)~~