FIGURE 5.

Effects of TIMPs and siRNATIMP-1 on proliferation or apoptosis induction of aHSCs pretreated with or without siRNAgp46. A, aHSCs transduced with 10 nm of siRNAgp46 were treated with recombinant rat TIMP-1 (50, 100 ng/ml) or mouse TIMP-2 (0. 5, 1.0 μg/ml), (the sequence is identical to rat TIMP-2 protein) in DMEM containing 2% FBS for 3 days. The number of cells stained with antibody against αSMA-Cy3 was counted in 40 random fields per slide. Data are presented as mean ± S.D. of triplicate samples. *, p < 0.05; **, p < 0.01. B, efficient knockdown of TIMP-1 was confirmed by quantitative RT-PCR at day 2 of transfection. C, MMP activity of the culture medium of aHSCs transduced with 10 nm of siRNATIMP-1 in serum-free DMEM for 24–72 h of transfection was assessed with fluorogenic MMP substrate. The fluorescence signal was measured using a microplate reader (340/485 nm), and results were expressed in percentages of MMP activity as mean ± S.D. *, p < 0.05 compared with siRNAGFP. D, cell numbers of aHSCs transduced with siRNATIMP-1 at 10 nm in DMEM with 2% FBS at day 3 of transfection were counted after they were stained with antibody against αSMA-Cy3 in 40 random fields per slide. Data are represented as mean ± S.D. of three independent experiments. **, p < 0.01 compared with siRNAGFP. E, aHSCs treated with siRNATIMP-1 in DMEM containing 2% FBS for 3 and 5 days were stained for apoptotic change. The apoptotic cells were counted in 40 random fields per slide. Data are mean ± S.D. of three independent experiments. **, p < 0.01 compared with siRNAGFP.