结缔组织生长因子和金属蛋白酶组织抑制因子-1 RNA干扰靶位的筛选

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。

RNA interference targets on connective tissue growth factor and tissue inhibitor of metalloproteinase-1 in vitro

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 mRNA 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.