TIMP-1基因缄默削弱SH-SY5Y细胞的增殖潜能并诱导其凋亡增强  

TIMP-1 Gene Silencing Impairs Proliferation and Induces Apoptosis of SH-SY5Y Cells

作　　者：陈敏霞[1] 郁卫东[2] 梁蓉[3] 杨丽君[1] 晁爽[1] 赵贺华[1] 郭静竹[1] 

机构地区：[1]北京大学人民医院儿科,北京100044 [2]北京大学人民医院临床分子生物学研究所,北京100044 [3]北京大学人民医院生殖医学中心,北京100044

出　　处：《中国生物化学与分子生物学报》2008年第11期1021-1028,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究发展规划(973计划;No.2001CB510303);国家自然科学基金(No.30570685)资助课题~~

摘　　要：前期研究发现,人基质金属蛋白酶组织抑制剂-1(tissue inhibitors of metalloproteinases-1,TIMP-1)在唐氏综合征(Down’s syndrome,DS)胎儿脑组织内表达下调.为了探讨TIMP-1表达下调参与DS脑病变发生的可能机制,本研究以人神经母细胞瘤细胞(SH-SY5Y)为模型,观察TIMP-1基因沉默后对其增殖和凋亡的影响.应用LipofectaminTM2000将TIMP-1特异性短发卡RNA(short hairpin RNA,shRNA)导入SH-SY5Y细胞,经嘌呤霉素筛选获得稳定表达TIMP-1-shRNA细胞株;应用RT-PCR、real-time PCR和Western印迹对干扰效率进行鉴定:与SH-SY5Y细胞相比,无论在mRNA水平还是蛋白水平,SH-SY5Y-TIMP-1-shRNA细胞中TIMP-1的表达显著下调(下调率接近100%).结果显示,已成功构建了TIMP-1基因沉默的SH-SY5Y细胞模型.在此基础上,通过MTT检测发现,TIMP-1基因沉默后SH-SY5Y细胞增殖减慢;流式细胞仪和荧光显微镜凋亡检测显示,TIMP-1基因沉默后SH-SY5Y细胞凋亡明显增加.这些研究结果表明,TIMP-1基因沉默能削弱SH-SY5Y细胞的增殖能力并增强SH-SY5Y的凋亡效应,提示TIMP-1可能是通过影响神经细胞的增殖和凋亡参与DS智力低下的发病过程.Tissue inhibitor of metalloproteinases-1 （TIMP-1） was reported to be down-regulated in the fetal brain with Down＇s syndrome （DS）. To understand the role of TIMP-1 in DS encephalopathy, as well as its effect on cell proliferation and apoptosis, we transfected SH-SYSY cells with TIMP-1-shRNAs, selected with puromycin and validated with real-time PCR and Western blotting, the obtained cells were named as SH- SY5Y-TIMP-1-shRNA. The levels of TIMP-1 mRNA and protein were almost 100% knocked down as compared to that of SH-SY5Y cells. In SH-SY5Y-TIMP-I-shRNA cells, we observed an impaired proliferation potential （MTT） and an induced apoptosis （fluorescence microscope and flow cytometry）. In summary, TIMP-1 gene silencing can effectively impair the proliferation and induce apoptosis in SH-SY5Y cells, suggesting that TIMP-1 down-regulation may be involved in DS pathogenesis .

关 键 词：TIMP-1 基因沉默 增殖 凋亡 SH-SY5Y细胞 

分 类 号：Q421[生物学—神经生物学] Q492.6[生物学—生理学]