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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。
Objective To screen RNA interference targets of connective tissue growth factor (CTGF) and tissue inhibitor of tissue inhibitor of metalloproteinase (TIMP-1), which can effectively inhibit hepatic fibrosis. Methods According to the sequence of rat CTGF and TIMP-1, three target sites of RNA interference were designed and double-stranded RNA (dsRNA) was synthesized. Each transfected or transfected with TGF-β1-stimulated rat liver (HSC). After 48h, the RNA was extracted and reverse transcribed into cDNA. The relative expression and inhibition rate of CTGF, TIMP-1 and procol-α1 mRNA were determined by fluorescence quantitative PCR. RIA method was used to detect the indexes of liver fibrosis in HSC culture supernatant. Results CTGF-3 group had the best inhibitory effect on CTGF mRNA and Procol-α1 mRNA (60.09% and 65.03%, respectively) when interfering with CTGF gene alone. CTGF- TIMP-1 and TIMP-1 mRNA inhibited the transcription of Procol-α1 mRNA (68.55% and 62.84%, respectively). In the presence of interference, CTGF-3 / TIMP -1-3 group had the strongest inhibitory effect on CTGF mRNA and TIMP-1 mRNA (the inhibitory rates were 76.60% and 79.03% respectively), whereas the inhibition of Procol-α1 mRNA by CTGF-2 / TIMP-1-3 group The strongest (inhibition rate of 85.79%). Conclusion The most effective target of RNA interference against CTGF and TIMP-1 was successfully screened out, and the combined interference was more effective than single interference.