【摘要】 目的研究抗转化生长因子β1锤头状核酶的胞外切割作用以及在肝星状细胞内的活性。方法运用计算机设计针对转化生长因子β1的锤头状核酶,制备核酶、失活核酶和靶RNA胞外转录物,进行核酶的胞外切割反应；构建编码核酶、失活核酶的真核表达载体,并转染入活化型肝星状细胞株内,分析核酶和TGFβ1的表达。结果该核酶在胞外具有良好特异的切割活性,在转染的肝星状细胞内高效表达且有效下调TGFβ1的表达；而失活核酶在胞外不具有切割活性,在胞内对TGFβ1表达的抑制作用轻微。结论此核酶在胞外具有特异切割活性,在肝星状细胞内抑制TGFβ1的表达,有望为肝纤维化的治疗提供新的手段。更多还原

【Abstract】 Objective To identify the activity of hammerhead ribozyme against transforming growth factor β1（TGFβ1） in a cell-free system and in activated hepatic stellate cells （HSCs）. Methods The ribozyme against TGFβ1 was designed with computer software. The transcripts of ribozyme, disabled ribozyme and target RNAs were prepared using the RiboMAX^TM large scale RNA production system. The in vitro cleavage reactions were performed through incubation of ³²plabeled target RNAs with ribozyme or disabled ribozyme in different conditions. The eukaryotic expression vector encoding ribozyme and disabled ribozyme were construeted, and then transfected into HSC-T6 cells which exhibited characteristics of activated HSCs. The intracellular activity of the ribozyme was determined by detecting the ribozyme, disabled ribozyme and the TGFβ1 expression. Results The ribozyme cleaved target RNAs into anticipated products effectively. As expected, the disabled ribozyme possessed no cleavage activity in vitro. Further study demonstrated that the ribozyme expressed efficiently and inhibited TGFβ1 expression in activated HSCs, while the disabled ri0bozyme displayed only a slight effect on TGFβ1 expression. Conclusion The ribozyme with perfect cleavage activity in the cell-free system used inhibited TGFβ1 expression effectively in activated HSCs. This ribozyme can provide a potential therapeutic approach for liver fibrosis.更多还原