SIRT1及其选择性剪接变异体SIRT1-ΔExon8在293T细胞衰老模型中的作用研究  

Effect of SIRT1 and its alternative splicing variant SIRT1-ΔExon8 on senescence model of 293T cells

作　　者：王玥[1] 杨小荣[1] 张策[1] 

机构地区：[1]山西医科大学基础医学院生理学系,太原030001

出　　处：《中国药物与临床》2015年第8期1064-1067,共4页Chinese Remedies & Clinics

基　　金：国家自然科学基金(31171023);国家青年科学基金(31000481);山西省高等学校优秀青年学术带头人支持计划(2011)

摘　　要：目的通过观察SIRT1全长(SIRT1-FL)及缺失第8外显子的SIRT1选择性剪接变异体(SIRT1-ΔExon8)在D-半乳糖诱导的293T细胞衰老模型中的表达变化,初步探讨SIRT1全长及SIRT1-ΔExon8对细胞衰老进程的影响。方法 1细胞计数kit-8(CCK8)法测定不同浓度D-半乳糖(0,5,10,15,20 g/L)对293T细胞活力的影响。2用含10 g/L的D-半乳糖达氏修正伊氏培养基作用于第3代293T细胞,继续培养至第6代,建立细胞衰老模型,β-半乳糖苷酶(SA-β-gal)染色观察衰老细胞阳性率。3实时聚合酶链反应(RT-PCR)检测诱导组与对照组细胞中SIRT1-FL及SIRT1-ΔExon8 m RNA的表达水平。结果 1CCK-8结果显示10 g/L的D-半乳糖能诱导细胞衰老且不引起细胞过度损伤。2诱导组衰老细胞阳性率(74.2±3.6)%高于对照组(7.7±1.4)%(P<0.05)。3RT-PCR结果显示诱导组SIRT1-FL和SIRT1-ΔExon8 m RNA表达水平分别较对照组降低了(78.26±0.05)%和(88.03±0.03)%(P<0.05)。结论 1SA-β-gal染色结果提示D-半乳糖诱导293T细胞衰老模型建立成功。2衰老细胞组的SIRT1-FL和SIRT1-ΔExon8m RNA表达水平较对照组均显著降低,提示SIRT1-FL及SIRT1-ΔExon8可能参与调控细胞衰老的进程。Objective To investigate the effect of silent mating type information regulation 2 homolog 1-full long （SIRT1-FL） and its alternative splicing variant SIRT1-AExon8 on the aging process of 293T cells by observing the expression changes of SIRT1 and SIRT1-AExon8 in the D-galactose （D-gal） induced senescence model of 293T cells. Methods （1）CeII Counting Kit-8 （CCK8） was used to determine the effect of D-gal on 293T cells viability at different concentrations （0, 5, 10, 15, 20 g/L）. （2）The 293T cells were cultured in DMEM high-glucose medium containing 10 g/L D-gal at the third passage and amplified to the sixth passage, and the senescence model of the 293T cells was established. β-galactosidase （SA-β-Gal） staining was used to determine the positive rate of senescent cells. （3）The mRNA expression levels of SIRT1 and SIRT1-AExon8 were measured in the 293T cells of the D-gal induced group and the control group by RT-PCR assay. Results （1）The CCK-8 assay showed that the 10 g/L D-gal could induce 293T cells senescence and avoid extensive injuries. （2）The positive rate of senescent cells in the D-gal induced group （74.2±3.6）% was higher than that in the control group （7.7±1.4）% （P〈0.05）. （3）The findings of RT-PCR showed that the mRNA expression levels of SIRTI-FL and SIRT1-AExon8 in the 293T ceils in the D-gal induced group were decreased by （78.26±0.05）% and （88.03±0.03）% compared with those in the control group, respectively （P〈0.05）. Conclusion （1）SA-β-gal staining shows that the D-gal can induce the senescence model of 293T cells successfully. （2）The mRNA expression levels of SIRT1-FL and SIRT1-AExon8 in the senescence cells group are significantly decreased compared with those in the control group, suggesting SIRT1-FL and SIRT1-AExon8 may be involved in the regulation of 293T cells senescence.

关 键 词：沉默信息调节蛋白质类 细胞衰老 半乳糖 

分 类 号：R339.38[医药卫生—人体生理学]