siRNA对HSC结缔组织生长因子的抑制作用

Inhibition Effect of Chemically Synthetic siRNA on Connective Tissue Growth Factor in HSC

目的研究化学合成的小分子干扰RNA (siRNA)对鼠肝星状细胞 (HSC)结缔组织生长因子 (CTGF)基因表达的抑制作用。方法针对大鼠CTGFmRNA 4 83、885和 94 6位点化学合成 3对 2 1核苷酸 (nt)siRNA ,分别以 5 0、10 0、2 0 0nmol/L浓度转染HSCT6 ,以空白及转染非特异siRNA作为对照 ,应用RT PCR检测CTGFmRNA表达 ,筛选出抑制效率最高的siRNA及其浓度 ;再转染HSCT6 ,抽提孵育 2 4、4 8、72h细胞总RNA及蛋白质 ,应用RT PCR和Westernblot检测CTGFmRNA及蛋白质表达。结果与对照相比 ,转染siRNA的HSCT6细胞CTGFmRNA表达水平明显降低 ,siRNA抑制效率在 5 0 ～2 0 0nmol/L范围呈浓度依赖性增高 ,以 4 83siRNA 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 4 83siRNA的HSCT6细胞 2 4 4 8、72hCTGFmRNA分别下调 (91± 2 ) %、(6 5± 3) % (P均 <0 .0 1)和 (10± 7) % (P =0 .0 6 34) ,蛋白分别下调 (86± 5 ) %、(94± 4 ) %和 (4 2± 9) % (P均 <0 .0 1)。结论化学合成 4 83siRNA能高效抑制HSC的CTGF基因表达 ,有效阻抑时间达 72h。

Objective To investigate the inhibition effect of chemically synthetic small interfering RNA (siRNA) on the expression of connective tissue growth factor (CTGF) gene in hepatic stellate cells (HSC) in rats. Methods Three pairs of 21 nucleotide(nt)siRNAs-directed rats’ CTGF mRNA 483, 885 and 946 targets were differently transfected into HSC T6 through oligofectamine package, untreated and transfected nonspecific siRNA HSC T6 as the blank control and the nonspecific siRNA control, respectively. The expression of CTGF at mRNA level in HSC T6 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The siRNA with consequent concentration which has the maximum inhibition rate was selected and transfected into HSC T6 again. Total RNA and protein of the cells after 24 h, 48 h and 72 h incubation were extracted, respectively. The expression of CTGF in mRNA and protein level were assessed by Western blot and RT-PCR. Results Three pairs of siRNAs reduced the mRNA level in HSC T6 in concentration-dependent manner within 50～200 nmol/L, and the maximum inhibition rate was found in 483 siRNA in 200 nmol/L; nonspecific siRNA could not affect CTGF mRNA expression in HSC T6. The expression of CTGF mRNA and protein was reduced by (91±2)%, (65±3)% (P<0.01), (10±7)% (P=0.0634) and (86±5)%, (94± 4)%,(42±9)% (P<0.01) at 24 h, 48 h and 72 h, respectively, in 483 siRNA-transfected HSC T6 compared with those in blank control. Conclusion Chemically synthetic 483 siRNA could significantly inhibit CTGF gene expression in HSCs and the effective suppression time is up to 72 h.It suggests chemically synthetic siRNA may be potential to prevent and treat liver fibrosis with excellent prospect of development.