小分子干扰RNA沉默结缔组织生长因子对鼠肝星状细胞培养激活的影响

Effect of silencing connective tissue growth factor by small interfering RNA on culture-activated rat hepatic stellate cells

目的研究化学合成小分子干扰RNA(siRNA)对大鼠原代肝星状细胞(HSC)结缔组织生长因子(CTGF)基因表达的阻抑作用及对HSC培养激活的影响。方法以胶原酶原位灌注消化加密度梯度离心法分离大鼠原代HSC,将siRNA以脂质体包裹,转染培养72h的原代HSC,设空白对照,收集孵育96h细胞,以Westernblot和免疫细胞化学染色法检测原代HSCCTGF及α-平滑肌肌动蛋白(α-SMA)基因表达。结果转染siRNA的原代HSCCTGF及α-SMA基因表达显著下调,与对照相比,CTGF蛋白下调(92±5)%(P<0.01);α-SMA蛋白表达分别下调(62±9)%(P<0.01,Westemblot方法)和(58±6)%(P<0.01,免疫细胞化学染色法)。结论siRNA能显著下调大鼠原代HSCCTGF基因表达,明显阻抑原代HSC培养激活,提示针对CTGF基因化学合成的siRNA具有防治肝纤维化的潜力。

Objective To investigate the inhibition of chemically synthetic small interfering RNA （siRNA） on the expression of connective tissue growth factor （CTGF） gene in rat primary hepatic stellate cells （HSCs） and the effect on the culture-activated rat HSCs.Methods Primary HSCs were isolated from normal SD rats by means of collagenase In situ perfusion digestion plus density gradient centrifugation, siRNA targeting CTGF was transfected into primary HSC which had been cultured for 72h by oligofectamine package, with untreated primary HSC as control. Cells after incubation with siRNA for 96h were collected. The expression of CTGF and α-SMA genes in primary HSCs was evaluated by Western blot and/or immunocytochemical staining. Results The levels of CTGF and α-SMA genes expression were markedly reduced in siRNA-transfected HSCs, the level of CTGF protein expression decreased by （92±5 ） % （ P 〈 0.01 ）, α-SMA protein （62± 9） % （ P 〈 0.01, Western blot） and （ 58 ± 6） % （ P 〈 0.01, immunocytochemical staining） compared with controls, respectively. Conclusion siRNA targeting CTGF could markedly down-regulate CTGF gene expression in primary HSCs, and significantly inhibit rats culture-activated HSCs. It suggests that chemically synthetic siRNA directing against CTGF gene may be of potential of prevention liver fibrosis.