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

发布时间：2018-03-05 19:40

本文选题：SIRT　切入点：青光眼小梁网细胞　出处：《四川大学学报(医学版)》2014年04期 　论文类型：期刊论文

【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。
[Abstract]:Objective to study the relationship between histone deacetylase (SIRT1) and repair ability and cell senescence of glaucoma trabecular meshwork (GTM) cells. Methods the expression of SIRT1 in normal trabecular meshwork cells and GTM was detected by RT-PCR and Western blot. Then the HTMM-GTM cells were divided into four groups: resveratrol group (0.5 渭 mol/L Res) and Control group (SIRT1 interference plasmid transfected cells) and Control group. The expression of SIRT1 was detected by Western blot. SA- 尾 -Gal senescence staining was used to detect the changes of cell senescence in each group after 3 days of continuous growth for 10 h or 32 h. Neutral comet electrophoresis was used to detect DNA double strand damage in cells treated with 1.33 mol / L H2O2 solution for 2 h. Western blot was used to detect the changes of 纬 -H2AX expression in cells treated with 1.33 mol / L H2O2 solution for 1 h or 2 h. Results the expression of SIRT1 was detected in both HTM and GTM cells. The expression level of sirt1 protein in HTM was higher than that in GTM group. It was found that the expression level of sirt1 protein was higher than that of HTM cell in Control group, microRNA34a group, SIRT1-ShRNA group, SA- 尾 -Gal senescence staining, and at the same time, the senescence degree of GTM cells was higher than that of HTM cell at the same time. In the same type of cells, the cells of SIRT1-ShRNA group showed senescence first, and with the prolongation of time, the senescence degree of the cells was the most serious after Res intervention for 24 hours. Neutral comet electrophoresis showed that the damage of double-stranded DNA in GTM group was more serious than that in HTM group. The results of 纬 -H2AX expression were consistent with those of neutral comet electrophoresis. Conclusion the down-regulation of SIRT1 expression activity may be one of the factors inducing glaucoma to increase HTM cells. The expression activity of SIRT1, Upregulated SIRT1 can promote the ability of DNA double strand damage repair in HTM cells, maintain the stability of cell genome, and then slow down cell senescence.
【作者单位】：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;中山大学中山眼科中心眼科学国家重点实验室;
【分类号】：R775

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