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MsrB1基因对H 2O 2诱导的人晶状体上皮细胞凋亡的保护作用
贾义
陈晋
作者及单位信息
·
DOI： 10.3760/cma.j.issn.2095-0160.2015.04.007
Role of MsrB1 gene in protection human lens epithelial cells against H 2O 2-induced apoptosis
Jia Yi
Chen Jin
Authors Info & Affiliations
Jia Yi
School of Biology and Bioengineering, Guiyang Medical University, Guiyang 550025, China
Chen Jin
·
DOI： 10.3760/cma.j.issn.2095-0160.2015.04.007
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摘要

背景氧化应激被认为是年龄相关性白内障发生的主要因素之一，研究表明蛋氨酸亚砜还原酶B1（MsrB1）对于保护晶状体细胞抵抗氧化应激具有重要作用，然而， MsrB1基因沉默对H 2O 2诱导的人晶状体上皮细胞（LECs）的凋亡影响的机制尚不十分清楚。

目的探讨 MsrB1基因沉默对H 2O 2诱导的人LECs凋亡及其细胞周期分布的影响。

方法人LECs细胞系SRA01/04在DMEM中进行培养，不同浓度的H 2O 2（200、400、600、800和1 000 μmol/L）加入培养基分别处理细胞12、24和36 h以选择最适浓度和作用时间。细胞分为正常对照组、MsrB1 siRNA转染组（将MsrB1 siRNA Lipofectamine 2000质粒转染入LECs）、H 2O 2诱导组（200 μmol/L作用LECs 24 h）和MsrB1 siRNA转染联合H 2O 2诱导组（转染MsrB1 siRNA脂质质粒后用H 2O 2处理细胞）。采用MTT法检测不同浓度H 2O 2诱导后人LECs 12、24和36 h细胞的存活率，并筛选H 2O 2处理作用最适的浓度和时间，用于进一步的细胞凋亡诱导实验；Real-time PCR法检测MsrB1 siRNA转染后24 h各组细胞中MsrB1 mRNA的相对表达水平；采用Hoechst 33528染色法观察各组细胞的细胞核形态变化；采用碘化丙啶（PI）染色法，用流式细胞仪检测细胞凋亡率和不同细胞周期的细胞比例。

结果正常对照组、H 2O 2诱导组、MsrB1 siRNA转染组细胞中MsrB1 mRNA的相对表达值为1.063±0.058、1.013±0.049和0.235±0.024，MsrB1 siRNA转染组细胞中MsrB1 mRNA的相对表达值明显低于正常对照组和H 2O 2诱导组，差异均有统计学意义（ t=31.744、35.807，均 P<0.001）。不同浓度H 2O 2处理后24 h细胞活力最低，以200 μmol/L H 2O 2处理后24 h为诱导人LECs凋亡的浓度和时间点。Hoechst 33528染色显示，H 2O 2诱导组可见部分细胞核收缩，MsrB1 siRNA转染组可见少数收缩变小的细胞核，MsrB1 siRNA转染联合H 2O 2诱导组收缩变小的细胞核多于H 2O 2诱导组。流式细胞术检测结果显示，正常对照组的凋亡细胞占（1.36±0.35）%，而MsrB1 siRNA转染组占（3.26±0.31）%，H 2O 2诱导组和MsrB1 siRNA转染联合H 2O 2诱导组细胞的凋亡率明显上升，分别为（5.13±0.59）%和（8.73±0.48）%。细胞周期检测结果表明，H 2O 2诱导组细胞周期阻滞在G 2/ M期。MsrB1 siRNA转染联合H 2O 2诱导组人LECs G 1期所占比例为（52.78±1.68）%，明显高于H 2O 2诱导组的（44.99±1.37）%，而G 2/M期细胞比例为（34.97±2.31）%，明显低于H 2O 2诱导组的（42.39±1.41）%，差异均有统计学意义（ t=6.224、-6.213，均 P<0.01）。

结论 MsrB1基因通过调节细胞周期的细胞分布减少H 2O 2诱导的人LECs的凋亡。

蛋氨酸亚砜还原酶;基因沉默／药物作用;人;晶状体;上皮细胞／药物作用;细胞凋亡;细胞周期
ABSTRACT

BackgroundOxidative stress is one of the major risk factors for age-related cataract, methionine sulfoxide reductase B1(MsrB1) has been shown to play an important role for lens cell function, resistance to oxidative stress. However, the mechanism of MsrB1 gene silencing on H 2O 2-induced human lens epithelial cells (LECs) apoptosis remains virtually unknown.

ObjectiveThis study attempted to investigate the role of MsrB1 gene in protecting human LECs against H 2O 2-induced damage.

MethodsHuman LECs cell line SRA01/04 cells were cultured in DMEM medium. Different concentrations (200, 400, 600, 800 and 1 000 μmol/L) of H 2O 2 were added to medium treat the cells for 12, 24 and 36 hours respectively to select the optimum inducing dose and time. The cells were divided into 4 groups. Lipofectamine 2000 containing MsrB1 siRNA was transfected into the cells in the MsrB1 siRNA transfected group, 200 μmol/L H 2O 2 treated the cells for 24 hours as the H 2O 2-induced group, and MsrB1 siRNA was transfected into the cells before H 2O 2 treatment as the MsrB1 siRNA transfected combined H 2O 2-induced group. The routinely cultured cells were as normal control group. Cell viability was detected by MTT assay. The relative expression of MsrB1 mRNA in the cells was analyzed by real-time PCR. Morphological changes of nuclei in the cells of different groups were observed using Hoechst 33528 staining under the fluorescence microscope. Cell apoptosis and cell cycle were quantified using flow cytometry.

ResultsThe relative expressing values of MsrB1 mRNA were 1.063±0.058, 1.013±0.049 and 0.235±0.024, and the expressing values of MsrB1 mRNA in the MsrB1 siRNA transfected group were significantly lower than those of the normal control group and H 2O 2-induced group ( t=31.744, 35.807, both at P<0.001). The cell viability was lowest 24 hours after 200 μmol/L H 2O 2 induced. Hoechst 33528 staining showed less contractive decrescent nuclei in the MsrB1 siRNA transfected group, and those in the H 2O 2-induced group and MsrB1 siRNA transfected combined H 2O 2-induced group were more. Flow cytometry revealed that the apoptotic rates in the normal control group, MsrB1 siRNA transfected group, H 2O 2-induced group and MsrB1 siRNA transfected combined H 2O 2-induced group were (1.36±0.35)%, (3.26±0.31)%, (5.13±0.59)% and (8.73±0.48)%. The cells distributed mainly in the G 2/M phase in the H 2O 2-induced group. The percentage of the cells in the G 1 phase of the MsrB1 siRNA transfected combined H 2O 2-induced group was (52.78±1.68)%, which was significantly higher than (44.99±1.37)% in the H 2O 2-induced group ( t=6.224, P<0.01), while the percentage of cells in the G 2/M phase also was significantly lower than that in the H 2O 2-induced group ([34.97±2.31]% versus [42.39±1.41 ] %) ( t=-6.213, P<0.01).

Conclusions MsrB1 gene plays a protecting role on human LECs against oxidative damage by decreasing cell apoptosis through regulating the cell cycle.

Methionine sulfoxide reductases;Gene silencing/drug effects;Humans;Lens;Epithelial cells/drug effets;Cell apoptosis;Cell cycle
Jia Yi, Email: mocdef.3ab61gnaixuoyiyaij
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引用本文

贾义,陈晋. MsrB1基因对H 2O 2诱导的人晶状体上皮细胞凋亡的保护作用 [J]. 中华实验眼科杂志,2015,33(4)：323-327.

DOI:10.3760/cma.j.issn.2095-0160.2015.04.007

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据报道，白内障已经成为人类致盲和视力残疾的主要因素，且对发展中国家造成了很大的经济和卫生负担 [ 1 , 2 ]。氧化应激被认为是年龄相关性白内障发生的主要因素之一 [ 3 ]。H 2O 2是晶状体和房水中与生理活动相关的氧化剂之一，白内障患者房水中的H 2O 2水平显著高于正常人，且体外研究发现，生理水平的H 2O 2会导致晶状体的混浊 [ 4 ]。晶状体蛋白中的蛋氨酸很容易被氧化为蛋氨酸亚砜 [ 5 ]，蛋氨酸亚砜还原酶(methionine sulfoxide reductases，Msrs)又可以将其还原为蛋氨酸 [ 6 ]。MsrB1是一种硒蛋白，定位于细胞核和细胞质中，且广泛分布于哺乳动物的不同组织中 [ 7 , 8 ]。有研究表明，MsrB1对于保护晶状体上皮细胞(lens epithelial cells, LECs)以抵抗氧化应激具有重要作用 [ 7 ]。虽然H 2O 2诱导的人LECs凋亡的机制已有报道 [ 9 ]，但 MsrB1基因沉默对H 2O 2诱导的人LECs细胞周期的影响及其机制尚不明确。本研究探讨H 2O 2对人LECs的损伤过程，并观察 MsrB1基因沉默对H 2O 2诱导的人LECs凋亡和细胞周期的影响。
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备注信息
A
贾义，Email： mocdef.3ab61gnaixuoyiyaij
B
贵州省科学技术基金项目 (黔科合J字[2014]2028号)
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