缺失第8外显子的SIRT1剪接体和SIRT1全长在293T细胞氧化损伤中的不同作用  

Exon 8-deficient SIRT1 splicing variant opposes SIRT1 full-length in regulating oxidative damage of 293T cells

作　　者：杨小荣[1] 高瑞君 李艳丽[1] 殷丽天[1] 张策[1] YANG Xiao-rong;GAO Rui-jun;LI Yan-li;YIN Li-tian;ZHANG Ce(Department of Physiology,Shanxi Medical University,and Key Laboratory of Cellular Physiology,Ministry of Education,Taiyuan 030001,China)

机构地区：[1]山西医科大学生理学系,细胞生理学教育部重点实验室,太原030001

出　　处：《解剖学报》2020年第6期855-860,共6页Acta Anatomica Sinica

基　　金：国家自然科学基金(31000481,31171023,81601167);山西省自然科学基金(201801D121316,2011011040-2);山西省“1331工程”重点学科建设计划(1331KSC);细胞生理学教育部重点实验室运行经费;山西省回国留学人员科研教研项目(HGKY2019054)。

摘　　要：目的探讨缺失第8外显子的沉默信息调节因子1(SIRT1)剪接变异体(SIRT1-ΔExon8)和SIRT1全长(SIRT1-FL)在氧化应激损伤中可能发挥的不同作用。方法在人胚肾293T细胞分别转染SIRT1-ΔExon8干扰质粒和SIRT1-FL过表达质粒,给予过氧化氢(H2O2)诱导氧化应激损伤模型,应用CCK-8检测细胞活力,乳酸脱氢酶(LDH)活性测定法检测细胞的损伤程度,流式细胞术检测细胞凋亡率和细胞内活性氧簇(ROS)水平,Real-time PCR法检测细胞SIRT1-ΔExon8和SIRT1-FL mRNA的水平。结果 H2O2(50~800μmol/L)可剂量依赖性地诱导细胞氧化应激损伤,包括细胞活力下降,LDH释放增加,细胞凋亡和胞内ROS含量增加,并引起SIRT1-FL mRNA水平降低和SIRT1-ΔExon8 mRNA表达增加;此外,过表达SIRT1-FL或干扰SIRT1-ΔExon8也可以部分抑制H2O2(400μmol/L)引起的细胞氧化应激损伤。结论 SIRT1-ΔExon8可能促进细胞的氧化应激损伤,而SIRT1-FL则发挥相反的作用。Objective To investigate the different effects of exon 8-deficient silent information regulator 1( SIRT1 splicing variant( SIRT1-ΔExon8) and SIRT1 full-length( SIRT1-FL) on cellular oxidative damage. Methods A human embryonic kidney 293 T cell line was infected with SIRT1-ΔExon8 shRNA expression vectors and SIRT1-FL overexpression vectors,respectively. Administration of hydrogen peroxide( H2O2) into 293 T cells was performed to induce oxidative stress injury including the changes of cell viability,lactate dehydrogenase( LDH) activity,apoptotic rate and reactive oxygen species( ROS) level as measured by cell counting kit 8( CCK-8) assay,LDH test,and flow cytometry. The mRNA levels of SIRT1-FL and SIRT1-ΔExon8 were determined by Real-time PCR. Results In a dose-dependent manner,50-800 μmol/L H2O2 induced oxidative damage including a decrease in cell viability,an increase in LDH release,cell apoptosis and ROS level;and also caused a decrease in SIRT1-FL mRNA level and an increase in SIRT1-ΔExon8 mRNA level. In addition,overexpression of SIRT1-FL or interference of SIRT1-ΔExon8 partly inhibited H2O2( 400 μmol/L)-induced stress injury.Conclusion SIRT1-ΔExon8 may promote cellular oxidative stress injury,while SIRT1-FL plays an opposite role.

关 键 词：过氧化氢 氧化应激 沉默信息调节因子1全长 沉默信息调节因子1-△Exon8 流式细胞术 

分 类 号：Q419[生物学—生理学] Q291