小干扰RNA抑制人端粒酶逆转录酶基因对恶性转化后骨髓间充质干细胞的影响  

Inhibitory Effect of siRNA Targeting hTERT Genes on the Transformed Mesenchymal Cells

作　　者：周晓岗[1] 李熙雷[1] 郭常安[1] 周健[1] 戴文达[1] 董健[1] 陈峥嵘[1] 

机构地区：[1]复旦大学附属中山医院骨科,上海200032

出　　处：《中国临床医学》2011年第3期269-274,共6页Chinese Journal of Clinical Medicine

基　　金：国家重点基础研究发展"973"计划(编号:2005CB522604);上海市科委重点基金(编号:06dj14001)

摘　　要：目的:探讨小干扰RNA(siRNA)抑制人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)表达对恶性转化后骨髓间充质干细胞(transformed mesenchymal stem cells,TMCs)增殖和在动物体内成瘤能力的影响。方法:构建针对hTERT的siRNA,通过脂质体转染的方法将hTERT-siRNA转染TMCs。实验组为3个浓度组,分别为50nmol/L、100nmol/L、200nmol/L,设阴性对照组和空白对照组。用噻唑蓝(MTT)法检测siRNA对TMCs生长增殖的影响。用Westernblot法检测各浓度组TMCs端粒酶的含量。逆转录-聚合酶链反应(RT-PCR)检测各浓度组hTERT mRNA含量,并用TRAP-ELISA法检测各浓度组TMCs端粒酶的活性。用BALA/C裸鼠体内实验检测siRNA转染后各组TMCs在动物体内成瘤能力的变化。结果:通过Western blot,发现不同浓度的hTERT-siRNA组TMCs的端粒酶蛋白含量逐渐减少,与siRNA的浓度呈负相关。50nmol/L、100nmol/L、200nmol/L组分别下降了46.2%、69.5%、91.5%;MTT法检测3个浓度组hTERT-siRNA对细胞的生长抑制率分别为2.74%、7.26%、10.23%。其中50nmol/L组和对照组之间差异无统计学意义,而100nmol/L组和200nmol/L组与阴性对照组之间差异有统计学意义。RT-PCR检测结果显示50nmol/L、100nmol/L、200nmol/L hTERT-siRNA转染组hTERT基因分别下降了19.2%、47.2%、86.4%。端粒重复序列扩增(TRAP)-酶联免疫吸附试验(ELISA)法检查发现,不同浓度的hTERT-siRNA对TMCs的端粒酶活性都产生抑制作用,3组分别下降了11.0%、42.9%、86.2%。100nmol/L组和对照组TMCs在裸鼠皮下形成肿瘤的时间相似,分别为14.8d、14.2d;200nmol/L组成瘤时间较晚,为17.3d。对照组形成的皮下肿瘤体积和质量分别为2.56cm3、1.30g;而100nmol/L和200nmol/L组肿瘤体积分别为0.64cm3、0.31cm3,质量分别为0.78g、0.58g,与对照组间相比差异有统计学意义。结论:siRNA可以下调hTERT基因的表达,减少TMCs细胞内端粒酶的含量,也降低了端粒酶的活性,从而增加了TMCs的凋�Objective：To investigate the effect of inhibiting human telomerase reverse transcriptase（hTERT） gene by small interfering RNA（siRNA） interference on the proliferation and the tumorigenicity ability of transformed mesenchymal stem cells（TMCs）.Methods：Small interference RNA（siRNA） targeting hTERT sequences were synthesized chemically in vitro.TMCs were transfected with three different concentrations of siRNA（50nmol/L,100nmol/L and 200nmol/L）.The proliferation activity of TMCs was determined using methylthiazol tetrazolium（MTT）.The expression of hTERT was detected by Reverse transcript polymerase chain reaction（RT-PCR）.The content and the activity of the telomerase were tested by western blot and telomeric repeat amplification protocolo（TRAP）-enzyme-linked immunosorbent assay（ELISA） staining.The ability of the tumorigenicity in nude mice of TMCs was also measured.Results：The content of temolerase of TMCs transfected by three different concentrations of siRNA（50nmol/L,100nmol/L and 200nmol/L） was obviously reduced than that of untransfected control group（46.2%,69.5% and 91.5%）.As detected by RT-PCR,mRNA level of hTERT in TMCs transfected with siRNA was reduced 19.2%,47.2% and 86.4% than that of control group,respectively.Telomerase activities of TMCs transfected by siRNA in all groups were also decreased.The proliferation and the abilities of tumorigenesis of TMCs transfected by siRNA in all groups were significantly reduced.Conclusions：hTERT expression in TMCs can be inhibited significantly with siRNA.Down-regulation of hTERT expression can cause the reduction of the proliferation of TMCs in vitro and inhibit their abilities of tumorigenesis in vivo.Telomerase is involved in the malignant transformation of MSCs.

关 键 词：骨髓间充质干细胞 恶性转化 小干扰RNA 端粒酶逆转录酶 

分 类 号：Q522[生物学—生物化学]