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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。
In this study, based on the verification of endogenous uPA gene expression in mouse testicular support cells TM4, three different siRNA sequences (si-uPA) were designed for uPA mRNA target sequences. TM4 cells were transiently transfected and the uPA gene Effective interference sequence. Effects of siRNA on uPA mRNA and protein expression in TM4 cells were observed by using the effective interference sequences. The results showed that the optimal transfection concentration of siRNA was 50 nmol / L. The expression of uPA mRNA and protein in TM4 cells transfected with three kinds of si-uPA were significantly lower than those in blank control group (P <0.05), and the most obvious effect was si-uPA1. The expression of uPA mRNA in transfected group was significantly lower than that in control group after 24 h transfection of si-uPA1, of which 100 nmol / L group showed the most obvious inhibitory effect, with the inhibition rate of 70%. With the extension of transfection time, uPA mRNA After 72 h of transfection, the expression levels of uPA mRNA in the three groups were 53.9%, 35.3% and 27.7% respectively (P <0.05). The study successfully screened uPA mRNA target sequence effective interference sequence, the inhibitory effect lasts up to 72 h; at the same time point, the inhibitory effect increases with the increase of transfection concentration, showing a good dose-effect relationship.