整合素连接激酶在重组结缔组织生长因子诱导大鼠肝星状细胞表型转化中的作用

目的研究整合素连接激酶(ILK)在重组结缔组织生长因子(rCTGF)诱导大鼠原代肝星状细胞(HSC)表型转化中的作用。方法应用胶原酶原位灌注+密度梯度离心法分离SD大鼠原代HSC,细胞贴壁培养24 h后以rCTGF处理,24 h后转染ILK小干扰RNA(siRNA)或对照siRNA,设rCTGF对照及空白对照。应用RT-PCR及Western Blot检测转染siRNA 24、48及72 h时HSCα平滑肌肌动蛋白(αSMA)、ILK及I型胶原基因表达水平变化。计量资料采用t检验。结果与空白对照相比,rCTGF处理可显著上调大鼠HSCαSMA、ILK蛋白及I型胶原mRNA表达。转染ILK siRNA可特异性抑制rCTGF诱导的ILK表达上调,与rCTGF对照相比,转染ILK siRNA 24、48及72 h时,HSC ILK蛋白表达分别下调72%±6%(t=21.39,P0.01)、87%±9%(t=68.25,P0.01)及47%±3%(t=18.25,P=0.003);αSMA蛋白及I型胶原mRNA表达分别下调5%±1%(t=2.52,P0.05)及6%±3%(t=1.63,P0.05)、31%±7%(t=34.77,P0.01)及20±5%(t=6.71,P0.05)和67%±8%(t=58.82,P0.01)及43%±6%(t=15.21,P0.01);转染对照siRNA时,HSC ILK、αSMA及I型胶原mRNA表达在相同时点无显著变化。结论 ILK可能部分介导了rCTGF诱导的大鼠HSC表型转化。

Role of integrin - linked kinase in phenotypic transformation of rat hepatic stellate cells induced by recombinant con- nective tissue growth factor

ObjectiveTo investigate the role of integrin-linked kinase （ILK） in the phenotypic transformation of rat＇s primary hepatic stellate cells （HSCs） induced by recombinant connective tissue growth factor （rCTGF）. MethodsPrimary HSCs were isolated from normal Sprague Dawley rats by in situ perfusion of collagenase and density gradient centrifugation. After 24 h of adherent cell culture, HSCs were treated with rCTGF for another 24 h before they were transfected with ILK small interfering RNA （siRNA） or control siRNA. An rCTGF control group （HSCs treated with only rCTGF） and a blank control group （HSCs treated with no rCTGF） were also set up. The gene expression levels of α-smooth muscle actin （αSMA）, ILK, and type I collagen in HSCs were measured by RT-PCR and Western blot at 24 h, 48 h, and 72 h after transfection with siRNA. The t-test was used for comparisons of measurement data. ResultsrCTGF significantly upregulated αSMA protein, ILK protein, and type I collagen mRNA in rat HSCs compared with the blank control group. Transfection of rat HSCs with ILK siRNA specifically inhibited the rCTGF-induced ILK upregulation. Compared with those in the rCTGF control group, the expression level of ILK protein in the ILK siRNA-transfected HSCs decreased by 72%±6% （t=21.39, P〈0.01） at 24 h, 87%±9% （t=68.25, P〈0.01） at 48 h, and 47%±3% （t=18.25, P=0.003） at 72 h; the expression levels of αSMA protein and type I collagen mRNA decreased by 5%±1% （t=2.52, P〉0.05） and 6%±3% （t=1.63, P〉0.05） at 24 h, 31%±7% （t=34.77, P〈0.01） and 20%±5% （t=6.71, P〈0.05） at 48 h, and 67%±8% （t=58.82, P〈0.01） and 43%±6% （t=15.21, P〈0.01） at 72 h. No changes were observed in the expression levels of ILK protein, αSMA protein, and type I collagen mRNA in HSCs transfected with control siRNA at the same time points. ConclusionILK may partly mediate the phenotypic transformation of rat HSCs induced by rCTGF.