抗血小板衍生生长因子小干扰RNA表达质粒构建及其对肝星状细胞的影响

Construction and evaluation of small interfering RNA plasmids targeting against B chain of platelet-derived growth factor mRNA and its effects on biological characters of hepatic stellate cells

目的研究靶向PDGF-B链的小干扰RNA(siRNA)对肝星状细胞(HSCs)细胞外基质分泌的影响。方法根据PDGF-B链序列设计合成siRNA序列,将siRNA序列插入pSilencer3.1-H1hygro质粒,酶切后测序鉴定;PDGF-B链siRNA表达质粒转染HSCs,潮霉素筛选阳性细胞株,RT-PCR和Westernblot分别检测PDGFmRNA和蛋白的表达;将各组筛选的阳性HSCs细胞株培养,MTT观察各组细胞增殖,放射免疫法检测HSCs细胞株上清液中透明质酸和Ⅲ型前胶原的表达。结果成功构建3种表达PDGF-B链siRNA表达质粒,RT-PCR和Westernblot结果表明各siRNA表达质粒阳性HSCs细胞PDGFmRNA和蛋白表达均有明显抑制。PDGF-B链siRNA表达质粒HSCs细胞组较对照组能明显降低HSCs增殖,下调透明质酸和Ⅲ型前胶原表达。结论靶向PDGF-B链的siRNA能降低HSCs增殖和分泌细胞外基质的能力,具有抗纤维化治疗的潜力。

Objective To construct small interfering RNAs(siRNAs) plasmids targeting against platelet-derived growth factor-B chain(PDGF-B) mRNA and evaluate their effects on the activity of hepatic stellate cells.Methods Expression plasmid vectors of anti-PDGF siRNA were constructed and subsequently transfected into HSC-T6 cells by rountine means.The positive clones were gained by hygromycin selection.The expression of PDGF was detected by RT-PCR and Western blot.The cell proliferation was determined through MTT.Contents of hyaluronic acid and type Ⅲ pro-collagen in the supernatants were determined by radioimmunoassay.Results The expression of anti-PDGF siRNA plasmids could markedly inhibit the expression of PDGF-B mRNA and protein.These siRNAs can inhibit the proliferation of HSCs and downregulate the secretion of hyaluronic acid and type Ⅲ pro-collagen.Conclusions The anti-PDGF siRNAs can inhibit the target RNA effectively and it may be a new therapy for liver fibrosis by inhibiting PDGF expression in HSCs by siRNA.