SIRT1对氧化应激下人小梁网细胞功能的影响

Effect of SIRT1 on cell function of human trabecular meshwork cell under oxidative stress

目的:通过在人小梁网细胞(human trabecular meshwork cell,HTMC)中过表达沉默信息调节因子2相关酶1(silent information regulator 1,SIRT1),探讨SIRT1对氧化应激下HTMC功能的影响。方法:将SIRT1过表达慢病毒和GFP阴性对照慢病毒按照最佳(multiplicity of infection,MOI)分别转染入HTMC,并用实时定量PCR法对SIRT1是否在细胞中过表达进行验证。实验分为以下4组:正常组、H2O2组、H2O2+Lv-SIRT1-OE(过表达)组、H2O2+Lv-GFP组,分别采用Transwell法和CCK8法检测氧化应激下HTMC的迁移能力和活性。两组间比较采用独立样本t检验。结果:在正常组、H2O2组、H2O2+Lv-SIRT1-OE组、H2O2+Lv-GFP组这4组中,Transwell实验结果分别为436±73、254±25、510±51、327±46,H2O2+Lv-SIRT1-OE组分别与H2O2组和H2O2+Lv-GFP组差异均有统计学意义(P<0.01)。CCK8法结果显示,H2O2+Lv-SIRT1-OE组分别与H2O2组和H2O2+Lv-GFP组相比差异均有统计学意义(P<0.01)。H2O2+Lv-SIRT1-OE组分别与H2O2组和阴性对照组(H2O2+Lv-GFP)相比,Bax表达水平明显下降,Bcl-2表达水平明显提高,差异均有统计学意义(P<0.01)。ROS活性氧测定显示H2O2+Lv-SIRT1-OE组比H2O2组的细胞活性氧水平显著降低(P<0.05)。结论:在HTMC中过表达SIRT1能有效降低氧化应激对HTMC迁移能力和活性的影响,从而对HTMC起到一定的保护作用,为后续研究SIRT1保护氧化应激下HTMC的调控机制打下基础。

Objective:To explore the effect of Silent Information Regulator 1(SIRT1)on cell function of human trabecular meshwork cell(HTMC)under oxidative stress by overexpressing SIRT1 in HTMC.Methods:This is an experiment research.HTMCs were transfected with SIRT1-ovexpressed lentivirus and GFP-negative control lentivirus(Lv-GFP)at the optimal multiplicity of infection(MOI).Real-time quantitative PCR was used to verify whether SIRT1 was overexpressed in HTMC.The following experiments were divided into four groups:normal control group,H2O2 group,H2O2+Lv-SIRT1-OE group,H2O2+Lv-GFP group.Cell migration was detected by transwell assay.Cell viability was detected by CCK8 assay.Student’s t-test was used for two groups.P<0.05 was set as statistical significance.Results:The number of migration per well of normal control group,H2O2 group,H2O2+Lv-SIRT1-OE group,H2O2+Lv-GFP group were 436±73,254±25,510±51,327±46,respectively.Compared with H2O2 group and H2O2+Lv-GFP group,transwell assay demonstrated that the number of migrations per well of H2O2+Lv-SIRT1-OE group significantly increased(P<0.01).Likewise,CCK8 assay indicated that cell viability of H2O2+Lv-SIRT1-OE group was higher than both of H2O2 group and H2O2+Lv-GFP group(P<0.01).Compared with H2O2+Lv-SIRT1-OE group and negative control group(H2O2+Lv-GFP),the expression level of Bax decreased significantly,and the expression level of Bcl-2 increased significantly(P<0.01).ROS assay showed that the ROS level in H2O2+Lv-SIRT1-OE group was significantly lower than that in H2O2 group(P<0.05).Conclusion:SIRT1 overexpressed in HTMC can effectively reduce the effect of oxidative stress on migration ability and proliferation activity of HTMC,which lays a foundation for further study on the regulatory mechanism of SIRT1 protecting HTMC under oxidative stress.