【摘要】 该研究在验证小鼠睾丸支持细胞TM4有内源性uPA基因表达的基础上,针对uPAmRNA靶序列设计三段不同的siRNA序列(si-uPA),通过瞬时转染TM4细胞,筛选确定uPA基因的有效干扰序列。将该有效干扰序列进行时效、量效实验,观察siRNA对TM4细胞uPA mRNA和蛋白表达的影响。结果显示,siRNA的最佳转染浓度为50 nmol/L。三种si-uPA转染TM4细胞后,uPA mRNA和蛋白表达量均较空白对照组明显下降(P<0.05),以si-uPA1作用最为明显。si-uPA1转染24 h后,转染组细胞uPA mRNA的表达均较对照组显著降低,其中100 nmol/L组抑制效果最为明显,抑制率达到70%;随转染时间的延长,uPA mRNA表达持续降低,转染72 h后,三组转染细胞uPA mRNA表达量分别为对照组的53.9%、35.3%和27.7%(P<0.05)。该研究成功筛选出针对uPA mRNA靶序列的有效干扰序列,抑制效应持续至72 h;同一时间点内,抑制效应随转染浓度的增加而增强,表现出良好的量效关系。更多还原

【Abstract】 The aim of our study is to analyze the suppressive effects of siRNA targeting uPA gene(si-uPA) in uPA expression in mice sertoli cell line(TM4 cells).3 different sequences of si-uPA were transfected into TM4 cells to screen the effective si-uPA.The inhibition of uPA expression was observed at various time points after transfection with the effective si-uPA in three different concentration(30,50,100 nmol/L).The results showed that the proper concentration of transfection for TM4 cells was 50 nmol/L.Among the three si-uPA,si-uPA1 had best suppressive effect.After transfection of si-uPA1 into TM4 cells,the decrease of uPA mRNA expression was ob-served in all three groups at 24 h,and the suppressive rate reached 70% in 100 nmol/L group,which was much ob-vious than those in 30 nmol/L and 50 nmol/L groups(P<0.05).The relative expression of uPA reduced steadily with the extension of transfection.At 72 h after transfection,the uPA mRNA in those three groups were 53.9%,35.3% and 27.7%,respectively,compared with those in blank control(P<0.05).In this study,the effective sequence of siRNA targeting uPA gene was screened from three candidates and the inhibition effect continued to 72 h.The sup-pressive effects of the same siRNA concentration showed no significant differences when detected at different time points,while variant concentration of siRNA inhibited the expression of uPA gene in a dose-dependent manner.更多还原