【摘要】 目的　设计、合成及筛选高效抗肝纤维化关键基因———结缔组织生长因子 (CTGF)的小分子干扰RNA(siRNA)。方法　应用RNA设计软件 ,模拟SD大鼠CTGFmRNA二级结构 ,设计并合成针对CTGFmRNA 483、885及946位点的三对 2 1核苷酸 (nt)siRNA ,转染肝星状细胞系 (HSCT6) ,以空白及转染非特异siRNA(与CTGFmRNA无同源性的 2 1ntsiRNA)作为对照 ,应用逆转录 聚合酶链反应 (RT PCR)检测HSCT6细胞CTGFmRNA表达。结果　与对照相比 ,转染siRNA的HSCT6细胞CTGFmRNA明显下调 ,在 5 0～ 2 0 0nmol/L范围 ,CTGFmRNA下调幅度呈浓度依赖性增加 ,以 483siRNA 2 0 0nmol/L最显著 ,转染非特异siRNA的HSCT6细胞CTGFmRNA表达水平无明显变化。转染 483siRNA的HSCT6细胞 2 4、48及 72hCTGFmRNA分别较对照下调 91%± 2 %、65 %± 3 % (t =78.82 ,3 7.5 3 ,P均 <0 .0 1)和 10 %± 7% (t =2 .5 5 ,P =0 .0 6) ,在 2 4～ 72h范围 ,CTGFmRNA下调幅度呈时间依赖性降低。结论　成功筛选到能高效阻抑CTGF表达的 483siRNA ,其有效阻抑时间约 72h ,有望通过介导CTGFmRNA剪切而抑制肝纤维化的发生与发展 ,可发展为新一代防治肝纤维化的核酸药物更多还原

【Abstract】 Objective To design, synthesize and screen high efficient small interfering RNA(siRNA) targeting to connective tissue growth factor (CTGF) in rat.Methods Three pair of 21 nucleotide siRNAs directed to CTGF mRNA 483,885 and 946 targets were designed and synthesized by utilizing RNA design software to simulate secondary structure of CTGF mRNA in SD rats. These siRNAs were differently transfected into HSC T6 (an active hepatic stellate cell strain in rat) by oligofectamine package, untreated and transfected unspecific siRNA HSC T6 as control. The expression of CTGF at mRNA level in HSC T6 was determined by reverse transcription polymerase chain reaction (RT PCR).Results Compared with bland controls, the expression of CTGF at mRNA level was markedly down regulated in siRNA transfected HSC T6, degree of down regulation in CTGF mRNA increased in a concentration dependent manner within 50 200nmol/L , especially 483 siRNA 200nmol/L, the expression of CTGF at mRNA level wasn’t found to have any significant changes in unspecific siRNA transfected HSC T6. The expression of CTGF at mRNA level down regulated 91%±2%,65%±3%( t = 78.82 , 37.53 , P <0.01 ) and 10%±7%( t = 2.55 , P = 0.06 ), respectively, in 483 siRNA transfected HSC T6 compared with blank control, extent of down regulation in CTGF mRNA decreased in a time dependent manner within 24 72hrs.Conclusions 483 siRNA, which can highly effectively inhibit expression of CTGF gene was screened successfully, its inhibition effect can maintain near 72hrs. It is hopeful that 483 siRNA will be a new nucleic acid drug that can inhibit the development of liver fibrosis through mediating CTGF mRNA degradation.更多还原