【摘要】 目的探讨靶向黏着斑激酶(FAK)基因的短发卡状RNA(shRNA)对大鼠肝星状细胞系HSC-T6活化与增殖的影响。方法分别设计2对有小发卡结构的DNA序列及1对非特异对照序列,构建重组质粒载体,经阳离子聚合物介导转染HSC-T6细胞。通过real-timePCR与Western blot进行筛选鉴定,改良MTT法观察细胞增殖,Western blot检测HSC活化的标志物α-SMA的表达。结果shRNA1、shRNA2均能抑制FAKmRNA和蛋白表达(P<0.05),其中shRNA2对FAK基因抑制率达76.82%,且可明显抑制HSC-T6细胞α-SMA的表达及细胞增殖。结论靶向FAK基因的shRNA可以抑制HSC-T6细胞的活化与增殖。更多还原

【Abstract】 Objective To investigate the role of small hairpin RNA(shRNA) targeting FAK gene on the activation and proliferation in rat hepatic stellate cells(HSC).Methods Two pairs of DNA sequences containing small hairpin structure and one pair of non-specific control sequence were cloned into the plasmid pGenesil-1.The recombinant plasmids were then transfected into HSC-T6 by cationic polymer gene transfection reagent.The expression of FAK was detected by real-time PCR and Western blot.The proliferation of HSC was detected by MTT assay.The expression of the marker of HSC activation,α-smooth muscle actin(α-SMA),was assessed by Western blot.Results The FAK shRNAs inhibited the expression of FAK at mRNA and protein level,and shRNA2 decreased FAK expression by 76.82% in mRNA level.FAK shRNAs significantly inhibited the proliferation of HSC and decrease the protein expression of α-SMA in rat HSC.Conclusion The specific FAK shRNA may have an inhibitory effect on the proliferation and activation in rat HSC.更多还原