沉默KLF4表达对人肝星状细胞HSC-LX2生物学行为的影响

目的 筛选有效沉默KLF4基因表达的siRNA,观察KLF4基因沉默对人肝星状细胞HSC-LX2生物学行为的影响.方法 设计并化学合成针对人KLF4基因的3对siRNA(siRNA1、siRNA2、siRNA3),转染人肝星状细胞HSC-LX2,以转染非特异siRNA作为阴性对照.采用实时荧光定量RT-PCR(Real-time RT-PCR)和Western blot方法分析KLF4 mRNA和蛋白的表达情况,筛选出沉默KLF4效果最好的1个siRNA,将其转染入HSC-LX2后,用MTT法检测KLF4沉默对HSC-LX2增殖的影响.采用ELISA法检测细胞培养上清中的转化生长因子1(transforming growth factor,TGF-1)、白介素1(interleukin-1,IL-1)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的表达情况.结果 Real-time RT-PCR和western blot均显示siRNA3沉默效果最好.故后续试验均采用siRNA3沉默KLF4的表达.MTT实验结果 显示,在转染siRNA3,12 h、24 h和48 h后,细胞活力出现明显下降.ELISA结果 显示,转染siRNA3,48 h以后,TGF-β1、TNF-α、IL-1β表达量也明显降低.结论 siRNA3可有效沉默KLF4基因表达,KLF4基因沉默可以抑制HSC-LX2的增殖,影响其生物学行为,使细胞中的促进胶原表达的三种细胞因子TGF-1、TNF-α以及IL-1表达量下降.

Effects of silencing KLF4 expression on biological behaviors of HSC-LX2 in vitro

Objective To screen the siRNA which can effectively silence KLF 4 expressions , and to observe the effects of KLF4 gene silence on biological behaviors of LX 2 in human hepatic stellate cells ( HSC-LX2 ) .Methods The three pairs of siRNAs (siRNA1,siRNA2,siRNA3)targeting at human KLF4 mRNA were designed and chemically synthesized , then which were transfected into HSC-LX2.The expression levels of KLF4 mRNA and protein were detected by Real-time fluorescent quantitative PCR ( Real-time RT-PCR) and Western Blot ,respectively ,with the nonspecific siRNA transfected into HSC-LX2 being regarded as negative control group .One of the siRNAs with the best effects of silencing KLF 4 expression was selected,then which was transfected into HSC-LX2, the effects of KLF4 silence on cell proliferation of HSCs were measured by MTT.Moreover the expression levels of transforming growth factor ( TGF-1 ) , interleukin-1 ( IL-1 ) , tumor necrosis factor-α( TNF-α) in supernatant of cell culture were detected by ELISA .Results The examination results by Real-time RT-PCR and Western Blot showed that the silencing effect of siRNA 3 was the best,thus,which was used in the subsequent experiments . MTT assay suggested that the cell vitalities were significantly decreased at 12h, 24h,48h after siRNA3 transfection, as compared with those in negative control group .ELISA assay showed the expression levels of TGF-1, TNF-α, IL-1 were obviously decreased at 48 h after transfection , as compared with those in negative control group .Conclusion The siRNA3 can effectively silence KLF4 expression,and KLF4 gene silence can inhibit the cell proliferation of HSC-LX2 and can affect its biological behaviors to decrease the expression levels of TGF-1, TNF-αand IL-1.