小干扰RNA技术下调视网膜母细胞瘤细胞株SO—Rb50中Survivin基因表达的研究  

To knockdown Survivin gene expression by siRNA in SO-Rb50 cells

作　　者：聂莉[1] 李永平[1] 聂栋[2] 彭展[1] 张卉颖[1] 张波[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广州510060 [2]北京大学深圳医院中心实验室

出　　处：《中华眼科杂志》2008年第10期906-911,共6页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目（30672276）;广州市科技攻关项目（3030902106005）

摘　　要：目的应用RNA干扰（RNAi）技术沉默视网膜母细胞瘤细胞株SO-Rb50中凋亡抑制因子Survivin的表达，观察其干扰效果及对细胞的影响。方法实验研究。体外化学法合成针对人Survivin基因的特异性小干扰RNA（siRNA）和对Survivin基因无沉默效果的阴性对照siRNA，利用转染试剂分别将siRNA转入SO-Rb50细胞株，通过半定量RT—PCR和Westernblot检测其对SO-Rb50细胞中Survivin基因和蛋白表达的抑制作用，四甲基偶氮唑盐（MTT）法检测不同浓度siRNA对细胞增殖抑制率，流式细胞仪检测转染后细胞凋亡率和细胞周期改变，荧光显微镜下观察细胞凋亡形态。对多组间数据比较采用单因素方差分析，处理组与对照组比较采用Dunnett-t检验。结果Survivin特异性siRNA转染细胞24h后Survivin mRNA和蛋白表达水平明显下调，对照组中其表达则无明显改变。MTT法结果显示Survivin特异性siRNA浓度为35、70、100nmol／L时均对SO-Rb50细胞增殖具有抑制作用（P〈0．05），且具有剂量依赖性。流式细胞仪检测发现Survivin特异性siRNA组出现凋亡亚二倍体峰，细胞被阻滞于G0／G1期，较对照组增加了8．11％，G2／M期细胞比例减少了5．75％，S期细胞仅减少了2．26％。荧光显微镜下可见部分细胞出现染色质浓缩和凋亡小体等典型的凋亡形态学变化。结论针对Survivin的RNA干扰技术可有效地下调SO-Rb50细胞中Survivin基因表达，进而抑制SO-Rb50细胞增殖和诱导其凋亡，为视网膜母细胞瘤的基因治疗提供了重要的途径。Objective To investigate cell proliferation and apoptosis status of human retinoblastoma cell line SO-Rb50 after knockdown of Survivin gene by means of small interfering RNA （siRNA）. Methods Survivin specific siRNA designed from the human gene sequence and nonsense siRNA（ as a negative control） was tranfected into SO-Rb50 cells. The inhibition of the expression of Survivin mRNA and protein levels were detected by reverse transcription-pelymerase chain reaction （RT-PCR） and Western blot. Proliferation inhibition rate of SO-Rb50 cells was analyzed by MTT assay. Apoptotic rate and cell cycle were analyzed by flow cytometry （FCM） and the apeptotic morphology was observed by fluorescent microscope. Results The expression of Survivin at both mRNA and protein level in specific siRNA group decreased significantly in comparison with untransfected group and nonsense siRNA group after 24 hours of transfection. The proliferation of SO-Rbw was inhibited in the Survivin specific siRNA group at the concentrations of 35,70 and 100 nmol/L（P 〈0.05 ）. Flow cytometry showed obvious apoptotic peak in Survivin specific group with an accumulation of cells in the G0/G1 phase and a decrease in G2/M phase and S phase. Typical apoptosis morphyology was also observed under fluorescent microscope. Conclusions Survivin specific siRNA could inhibit SO-Rb50 cell proliferation and induced apoptosis by knockdown of Survivin gene. Our data suggests that the use of Survivin-specific siRNA deserves further investigation as a novel approach to retinoblastoma therapy.

关 键 词：视网膜母细胞瘤 细胞系 肿瘤 RNA 小分子干扰 细胞凋亡 微管相关蛋白质类 

分 类 号：R686[医药卫生—骨科学]