针对不同靶位点的siRNA对人晶状体上皮细胞NF-κB P65表达的抑制作用  

Inhibition of small interference RNA targeting nuclear factor-κB P65 on expression of nuclear factor-κB in human lens epithelial cells

作　　者：张丽娜[1] 赵桂秋[1] 梁涛[1] 王青[1] 杨姗姗[1] 

机构地区：[1]青岛大学医学院附属医院眼科,在读博士研究生266003

出　　处：《眼科研究》2008年第8期586-589,共4页Chinese Ophthalmic Research

基　　金：山东省科委基金资助(2004GG3202023)

摘　　要：目的探讨针对核因子-κB(NF-κB)P65亚基的小干扰RNA(siRNA)对晶状体上皮细胞(LECs)NF-κB表达的影响。方法设计并合成3条NF-κBsiRNA,1条阴性对照siRNA。实验分为6组,A组为正常对照组,常规体外培养HLE-B3细胞;B组为肿瘤坏死因子-α(TNF-α)刺激组;C、D、E、F组为转染组,分别转染4条siRNA,24h后加入含TNF-α的培养基培养。免疫印迹法、实时荧光定量PCR法分别检测NF-κBP65蛋白与mRNA的表达。结果B组与A组比较,NF-κBP65表达明显增加,差异有统计学意义(P<0.01)。C组、D组与B组比较,NF-κB表达明显减少,差异有统计学意义(P<0.01)。结论特异性siRNA可以明显降低HLE-B3细胞株NF-κBP65蛋白及mRNA的表达,为RNA干扰治疗外伤性白内障奠定了基础。Objective Nuclear factor-kappaB（NF-κB）is a key mediator of inflammatory response.In the ophthalmology,NF-κB has been implicated in causing cataract after trauma.Small interference RNA（siRNA）targeted to human nuclear factor-κB（NF-κB）P65 was designed and the effect of NF-κB P65 siRNA on expression of NF-κB in human lens epithelial cells（HLE-B3）in vitro was evaluated in this study.Methods Three siRNAs（P1,P2,P3）specific for human NF-κB P65 and one scramble siRNA（P4）were designed and synthesized.The experiment included 6 groups.Group A：HLE-B3 cells were cultured in vitro in DMEM.Group B：HLE-B3 cells were cultured in DMEM with 20 ng/L tumor necrosis factor-α（TNF-α）for 24 hours.Group C,D,E,F：HLE-B3 cells were cultured in DMEM with 20 ng/mL TNF-α for 24 hours after transfected with P1,P2,P3,P4 respectively.NF-κB P65 protein and mRNA were detected by Western blot and Real-time PCR respectively.Results The expression of NF-κB P65 protein and mRNA in HLE-B3 cells was statistically significantly different in 6 groups（Fprotein=96.18,P〈0.01;FmRNA=613.88,P〈0.01）and was significantly increased in group B in comparison with group A（qprotein=14.355 9,P〈0.01;qmRNA=41.519 6,P〈0.01）.However,the expression levels of NF-κB P65 protein and mRNA were significantly decreased in group C and D in comparison with group B,but there were no significant difference in expression levels of NF-κB P65 protein and mRNA between group E,F and B.Conclusion Delivery of siRNA targeted to human nuclear factor-κB can be used in vitro to reduce the level of the specific protein and mRNA product（NF-κB P65）in HLE-B3 cells.This work lay a basis for the treatment of traumatic cataract by use of RNA interference.

关 键 词：RNA干扰 核因子-ΚB 晶状体上皮细胞 肿瘤坏死因子-α 眼外伤 白内障 

分 类 号：R776.1[医药卫生—眼科]