【摘要】 目的研究化学合成纤溶酶原激活物抑制剂-1(PAI-1)小干扰RNA(siRNA)对肝星状细胞(HSC)PAI-1基因表达及生物学特性的影响。方法将化学合成抗PAI-1 siRNA以Lipofectamine包裹,转染HSC-T6细胞,设阴性对照和空白对照,抽提细胞总RNA及蛋白质,收集培养上清液,应用逆转录-聚合酶链反应和免疫细胞荧光法检测细胞PAI-1表达,应用MTT法和流式细胞仪检测细胞增殖和细胞周期变化,应用酶联免疫吸附法检测培养上清液中Ⅰ、Ⅲ型胶原含量。结果转染siRNA的HSC- T6细胞PAI-1基因表达水平明显下调,HSC-T6细胞增殖抑制明显,48 h和72 h时培养上清液中Ⅰ型胶原表达水平显著减少,分别比阴性对照下降(20.71±8.40)%和(37.97±6.40)%(F=42.69,P= 0.0001),Ⅲ型胶原含量于72 h时降低(35.98±4.60)%(F=105.52,P=0.0001)。结论化学合成抗PAI-1 siRNA能高效抑制HSC-T6 PAI-1表达,抑制HSC-T6细胞增殖,显著减少Ⅰ、Ⅲ型胶原的合成和分泌,具有预防及治疗肝纤维化的潜力。更多还原

【Abstract】 Objective To investigate the effect of synthetic small interfering RNA (siRNA) targe- ting plasminogen activator inhibitor-1 (PAI-1) on the biological characters of hepatic stellate cells (HSCs).Methods Synthetic siRNA targeting PAI-1 was transfected into HSC-T6 by lipofectamine package.Negative siRNA transfection and no transfection were used as negative control and blank con- trol respectively.After incubation with siRNA,total RNA and protein of HSC-T6 cells were extracted. The expression of PAI-1 gene was detected by reverse transcription-polymerase chain reaction and immuno- cytofluorescent.The proliferation of HSC-T6 was determined using MTT assay.HSC-T6 cycle and apoptosis were measured by flow cytometry.Content of typeⅠandⅢcollagen in the supernatants were determined by enzyme linked immunosorbent assay.Results The expressions of PAI-1 mRNA and pro- tein were markedly down-regulated in siRNA-transfected HSC-T6.The proliferation of HSC-T6 was inhibited by transfection of siRNA.The level of typeⅠcollagen in the supernatants decreased by (20.71±8.40)% and (37.97±6.40)%,repectively(F=42.69,P=0.0001) compared to those in the negative control at 48 and 72 hours.The level of typeⅢcollagen decreased by (35.98±4.60)% (F=105.52,P= 0.0001) at 72 hours.Conclusion Inhibition of PA-1 by siRNA may have a potential effect in prevention and treatment of hepatic fibrosis by inhibiting proliferation,promoting apoptosis of stellate cells.Therefore,it decreases the synthesis and the secretions of typeⅠandⅢcollagen.更多还原