sirt2基因高表达对人脐带间充质干细胞衰老的抑制作用  

Inhibition of sirt2 overexpression on aging of human umbilical cord mesenchymal stem cells

作　　者：马珊珊[1] 崔渊博[2] 姚宁[1] 邢衢 韩康[1] 宋及时 张彦婷[1] 关方霞[1,3] 

机构地区：[1]郑州大学生命科学学院干细胞研究室,郑州450001 [2]郑州大学附属郑州中心医院转化医学中心,郑州450007 [3]郑州大学第一附属医院干细胞研究室,郑州450052

出　　处：《郑州大学学报（医学版）》2016年第1期10-14,共5页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目U1404313;81471306;河南省高校科技创新团队基金资助项目15IRTSTHN022;河南省科技创新人才计划基金资助项目154200510008

摘　　要：目的:观察sirt2基因高表达对人脐带间充质干细胞(h UC-MSCs)衰老的影响。方法:扩增人sirt2 ORF序列,利用腺病毒载体构建sirt2基因高表达的重组腺病毒(p Ad-sirt2),空斑法测定病毒滴度;感染h UC-MSCs,倒置荧光显微镜下观察荧光表达情况;实验分为P3组(第3代h UC-MSCs)、P15组(第15代h UC-MSCs)和感染组(用p Ad-sirt2腺病毒感染第15代h UC-MSCs),CCK-8检测细胞增殖情况,流式细胞术分析细胞周期变化,β-半乳糖苷酶染色后检测阳性细胞率。结果:成功制备p Ad-sirt2重组腺病毒,病毒滴度为1.8×1011pfu/L;荧光观察证实Sirt2蛋白在h UC-MSCs中有效表达;与P15组相比,sirt2高表达可促进h UC-MSCs增殖,并将细胞周期阻滞在S期,β-半乳糖苷酶阳性细胞率降低(P均<0.05)。结论:sirt2基因高表达可促进h UC-MSCs增殖,延长细胞周期,并有效抑制h UC-MSCs的衰老。Aim： To construct a recombinant adenovirus containing sirt2 gene and to explore the impact of sirt2 overexpression on the aging of human umbilical cord mesenchymal stem cells（ h UC-MSCs）. Methods： Human sirt2 ORF was generated by RT-PCR and the recombinant adenovirus（ p Ad-sirt2） was constructed. The viral titer was determined by air-dilution method. After being transfected into h UC-MSCs,the green fluorescence was observed under fluorescence microscope.h UC-MSCs were allocated into P3 group（ P3 h UC-MSCs）,P15 group（ P15 h UC-MSCs） and transfected group（ transfected h UC-MSCs by p Ad-sirt2）. CCK-8 assay was performed to detect the proliferation of h UC-MSCs and flow cytometry,to analyze the cell cycle. SA-β-Gal was used to detect the cell senescence. Results： The recombinant adenovirus was constructed and the titer was 1. 8 × 1011 pfu / L. After infection by p Ad-sirt2,P15 h UC-MSCs expressed green fluorescence protein clearly under the fluorescence microscope. Compared with P15 group,sirt2 overexpression could promote the cell proliferation,induced more cells at S phase,and decrease the positive rate of SA-β-Gal（ P〈0. 05）. Conclusion： sirt2 overexpression could promote h UC-MSCs proliferation,extend cell cycle,and inhibit cell senescence.

关 键 词：sirt2 腺病毒 人脐带间充质干细胞 衰老 

分 类 号：Q291[生物学—细胞生物学]