【摘要】 目的 :探讨RNA干扰技术(RNAi)靶向抑制卷曲蛋白(Frizzled)受体对人瘢痕疙瘩成纤维细胞(KFB)的抑制作用。方法:将针对人Frizzled受体基因设计合成的3对特异性小干扰RNA(si RNA)分别转染体外培养的人KFB,通过实时荧光定量聚合酶链式反应(q PCR)和western blot筛选出干扰人KFB Frizzled受体基因表达的最佳si RNA,进而转染人KFB,并采用q PCR及western blot检测Frizzled受体及相关基因和蛋白表达;采用细胞增殖检测试剂(CCK)-8检测细胞增殖情况。结果:si RNA303具有最佳的Frizzled受体m RNA和蛋白质抑制效率,Frizzled受体si RNA转染后,KFB的Frizzled受体、Wnt2、糖原合酶激酶(GSK)3β和D型细胞周期蛋白(cyclin D)1 m RNA及蛋白表达均下降。p-GSK3β蛋白表达下降,KFB生长受抑制程度增大,增殖减慢,且随着培养时间的延长,细胞倍增时间明显延迟。结论:转染靶向Frizzled受体的si RNA可有效敲低该基因在KFB内的表达,从而抑制成纤维细胞增殖。更多还原

【Abstract】 Objective: To study the suppressive effects of knocking down the expression of Frizzled receptor with small interfering RNA(si RNA) on keloid formation in vitro. Method: Human keloid fibroblasts(KFB) were transfected by 3 pairs of specific si RNA, which were designed and synthesized against the human Frizzled receptor gene. Quantitative real-time polymerase chain reaction(q PCR) and western blot were used to identify the most effective si RNA inhibiting expression of Frizzled receptor in human KFB. Following transfection with the most effective si RNA, expression levels of related genes and proteins were assessed using q PCR and western blot, respectively. Cell proliferation was evaluated using CCK-8 assay. Results: The303 si RNA displayed the most potent inhibition on both gene and protein expression. Transfection with 303 si RNA decreased ex pression levels of both m RNA and proteins of Frizzled receptor, Wnt2, GSK3β and cyclin D1 in addition to a reduction in expression of p-GSK3β protein. The extent of inhibition on KFB growth positively correlated with the treatment time. Cell doubling time was significantly delayed. Conclusions: Transfection with target si RNA target can knockdown Frizzled receptor gene, leading to inhibition of KFB growth.更多还原