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，Transwel l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 signifificance. 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.
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 signi?cance. 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.
