SIRT1增强青光眼小梁网细胞DSBs修复能力及抗细胞衰老的研究  

SIRT1 Promote GTM Cell DSBs Repair and Resist Cellular Senescence

作　　者：任朋亮[1] 范雪娇[1] 杨晓龙[1] 刘明佳[1] 刘戟[1] 黄晶晶[2] 

机构地区：[1]四川大学华西基础医学与法医学院生物化学与分子生物学教研室,成都610041 [2]中山大学中山眼科中心眼科学国家重点实验室,广州510060

出　　处：《四川大学学报（医学版）》2014年第4期572-577,共6页Journal of Sichuan University(Medical Sciences)

摘　　要：目的研究Ⅲ类组蛋白去乙酰化酶SIRT1与青光眼小梁网细胞(GTM)DNA双链损伤(DSBs)修复能力及细胞衰老之间的关系。方法首先通过RT-PCR和Western blot检测正常小梁网细胞(HTM)与GTM之间SIRT1表达的差异;然后将HTM、GTM细胞分别分为4组,白藜芦醇组(0.5μmol/L Res干预24h)、SIRT1-ShRNA组(构建成功的SIRT1干扰质粒转染细胞)、microRNA34a组(构建成功的microRNA34a表达质粒转染细胞)以及Control组,通过Western blot检测各组细胞中SIRT1表达水平的差异;SA-β-Gal衰老染色检测连续生长10h、32h、3d、6d后各组细胞的衰老变化;中性彗星电泳检测经1.33mol/L H2O2液处理0h、2h时各组细胞中DNA双链损伤情况;Western blot检测1.33mol/L H2O2液处理0h、1h、2h后,各组细胞中γ-H2AX表达的变化情况。结果 HTM及GTM细胞中均存在SIRT1的表达,且HTM中的表达要高于GTM;HTM、GTM细胞各组内比较发现,SIRT1蛋白的表达水平呈Res组>Control组>microRNA34a组>SIRT1-ShRNA组趋势;SA-β-Gal衰老染色则发现在相同时间点,GTM细胞的衰老程度均高于HTM细胞,而在同一类细胞中,SIRT1-ShRNA组细胞最先表现出衰老,且随着时间的延长,其衰老程度也最为严重,Res干预细胞24h后,能明显延缓细胞衰老。中性彗星电泳检测表明经氧化剂处理后,GTM组双链DNA的损伤情况比HTM组严重,并呈现出SIRT1-ShRNA组>microRNA34a组>Control组>Res组的趋势,γ-H2AX表达量的检测结果与中性彗星电泳的结果一致。结论 SIRT1表达活性的下调可能是诱发青光眼的因素之一;Res能显著增强HTM细胞中SIRT1的表达活性,上调的SIRT1能促进HTM细胞DNA双链损伤修复的能力,维持细胞基因组的稳定性,进而减缓细胞的衰老。Objective To study the relationship between SIRT1and glaucoma trabecular meshwork cell（GTM）cell on DNA double-strand breaks（DSBs）repair capability and resist cellular senescence.Methods The expressions of SIRT1in GTM and normal trabecular meshwork（HTM）cell detected by RT-RCR and Western blot;HTM and GTM cells divided into four groups separately：Res group（treat cells with 0.5μmol/L Resveratrol for 24h）,SIRT1-ShRNA group（cells infected with recombinant SIRT1-ShRNA）,microRNA34agroup（cells infected with recombinant microRNA34a）and control group.The expression level of SIRT1in groups was detected by Western blot.SA-β-Gal staining was applied to each group of cells at 10h,32h,3dand 6dto evaluate the senescence of the cells.DSBs and the expression ofγ-H2AX after treated with 1.33mol/L H2O2at 0h,1h,2h were detected by comet electrophoresis and Western blot.Results The expression of SIRT1were observed in both HTM and GTM cells,but the expression level in HTM was higher than that of GTM cells.have the ability to express SIRT1,however the expression of SIRT1was lower than HTM.Expression levels of SIRT1presented following treads：Res＆gt;Control＆gt;microRNA34a＆gt;SIRT1-ShRNA.The dgree of senescence in GTM was higher than that in HTM cells when detected at the same time point with SA-β-Gal staining.In the same cell line,the signs of senescence were appeared firstly and seriously in the cells treated with SIRT1-ShRNA in a timedependent manner.Differently,after 24htreatment with Res,the degree of senescence was decreased.The DSBs in GTM group was more than that of HTM group after treatment with oxidant when detected with Comet Electrophoresis and the the trends of the change was SIRT1-ShRNA＆gt;microRNA34a＆gt;Control＆gt;Res.The simillar results also observed in the expression ofγ-H2AX.Conclusion SIRT1may be useful in predicting the development and prognosis of glaucoma;Res promotes the expression of SIRT1significantly,and the SIRT1may protects GTM from oxidative stress-induced DSBs,agin

关 键 词：SIRT1 青光眼小梁网细胞 氧化应激 DNA双链损伤修复 细胞衰老 

分 类 号：R775[医药卫生—眼科]