结缔组织生长因子和金属蛋白酶组织抑制因子-1RNA干扰靶位的筛选

摘要

Objective To screen RNA interference (RNAi)targets on connective tissue growth factor(CTGF)and tissue inhibitor of metalloproteinase-1 (TIMP-1)in vitro.Methods Three candidates RNAi targets for rat's CTGF and TIMP-1 gene were designed and the double-stranded RNA (dsRNA)were synthesized.Then, single or different combined dsRNAs were transfected into rat hepatic stellate cells (HSCs)activated by TGF-β1.The procol-α1,CTGF and TIMP-1 mRNA were detected by fluorescent quantitative PCR,and hepatic fibrosis indexes in HSC supernatant were detected by radioimmunoassay.Results The CTGF mRNA and procol-α1 mRNA were inhibited at 68.09％ and 65.03％,respectively in CTGF-3 group when single RNAi targeted on CTGF gene was used,and the TIMP-1 mRNA and procol-α1 mRNA were inhibited at 68.55％ and 62.84％,respectively in TIMP-1-3 group when single RNAi targeted on TIMP-1 gene was used;When double RNAi targeted on CTGF gene and TIMP-1 gene were used,the CTGF mRNA and TIMP-1 mRNA were inhibited at 76.60％ and 79.03％,respectively in CTGF-3/TIMP-1-3 group,while CTGF-2/TIMP-1-3 group had the most effective effect on inhibiting procol-α1 mR-NA with inhibition rate of 85.79％.Conclusion The most effective RNAi targets on CTGF and TIMP-1 were selected successfully and the double RNAi targeting on CTGF and TIMP-1 were stronger than any single RNAi.%目的 筛选能有效抑制肝纤维化形成的结缔组织生长因子(CTGF)和金属蛋白酶组织抑制因子-1(TIMP-1)的RNA干扰靶位.方法 根据大鼠CTGF 和TIMP-1基因序列,分别设计3个RNA干扰候选靶位,化学合成双链RNA(dsRNA),各自转染或不同组合后转染经TGF-β1刺激活化的大鼠肝星状细胞(HSC),48h后抽提RNA并逆转录成cDNA,采用荧光定量PCR法测定并计算CTGF和TIMP-1、Ⅰ型前胶原(procol-α1)mRNA的相对表达量与抑制率,采用RIA法检测 HSC培养上清液肝纤维化指标.结果 在进行单独针对CTGF基因干扰时,CTGF-3组对CTGF mRNA及其下游产物Procol-α1 mRNA的转录抑制作用最佳(抑制率分别达68.09%与65.03%),在单独针对TIMP-1基因干扰时,TIMP-1-3组对TIMP-1 mRNA及其下游产物Procol-α1 mRNA的转录抑制作用最佳(抑制率分别达68.55%与62.84%);在联合干扰时,CTGF-3/TIMP-1-3组联合对CTGF mRNA和TIMP-1 mRNA的抑制最强(抑制率分别达76.60%与79.03%),而以CTGF-2/TIMP-1-3组联合对Procol-α1 mRNA的抑制作用最强(抑制率达85.79%).结论 成功筛选出针对CTGF和TIMP-1最有效的RNA干扰靶位,联合干扰较单独干扰效果更强.