目的 观察特异性miRNA-200b抑制剂对肝星状细胞增殖、活化、胶原合成的影响.方法 设计并合成miR-200b抑制剂,利用脂质体将miRNA-200b抑制剂转入肝星状细胞中,培养48 h后,收集肝星状细胞和上清液,采用qRT-PCR探针的方法检测miR-200b的表达水平、Westernblotting检测细胞α-平滑肌肌动蛋白(α-SMA)表达、噻唑蓝(MTT)法检测细胞增殖活性和放射免疫法检测培养上清液中Ⅲ型前胶原和透明质酸含量.结果 与阴性对照组相比,miRNA-200b抑制剂转染48 h后HSC-T6细胞miR-200b组表达下调82％;α-SMA蛋白表达降低(19±3)％(P＜0.05);细胞增殖活性降低(33±5)％(P＜0.01);培养上清中Ⅲ型前胶原和透明质酸含量分别降低(35±4)％和(31±2)％(均P ＜0.01).结论 miRNA-200b抑制剂下调HSC-T6细胞中miR-200b表达,抑制肝星状细胞增殖与活化,减少细胞外基质的合成和分泌.%Objective To investigate the effect of miRNA-200b-specific inhibitor on hepatic stellate cells(HSCs) activation,proliferation,and extracellular matrix production.Methods The miRNA-200b-specific inhibitors were designed,synthesized,and transfected into HSCs with lipofectamine 2000.The supernatant and HSCs were collected after incubation for 48 h.The expression of miR-200b was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR).The expression ofα-smooth muscle actin (oα-SMA) protein in HSCs was detected by Western blotting.The cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) method.Contents of type Ⅲ procollagen and hyaluronic acid in supernatant were determined by radioimmunoassay.Results Compared to the control group,miRNA-200b expression was decreased in the miRNA-200b inhibitor group by 82％ (P ＜ 0.01),α-SMA protein expression was reduced in the miRNA-200b inhibitor group by (19 ± 3) ％ (P ＜ 0.05),and the activity of HSCs proliferation was reduced by(33 ± 5)％ (P ＜0.01),and the contents of type Ⅲ procollagen and hyaluronic acid in supernatant were reduced in miRNA-200b inhibitor group by (35 ± 4)％ and (31 ± 2)％,respectively(P ＜0.01).Conclusions The miRNA-200b-specific inhibitor could significantly reduce the expression of miRNA-200b,and inhibit HSC proliferation,activation,and extracellular matrix production.
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