siRNA干扰小鼠睾丸支持细胞μPA表达的初步研究

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TM4fi细胞uPAmRNA和蛋白表达的影响。结果显示，siRNA的最佳转染浓度为50nmol／L。三种si—uPA转染TM4细胞后，μPAmRNA和蛋白表达量均较空白对照组明显下降（P〈0．05），以si—μPAl作用最为明显。si—μPAl转染24h后，转染组细胞μPAmRNA的表达均较对照组显著降低，其中100nmol／L组抑制效果最为明显，抑制率达到70％；随转染时间的延长，μPAmRNA表达持续降低，转染72h后，三组转染细胞μPAmRNA表达量分别为对照组的153．9％、35．3％和27．7％（P〈0．05）。该研究成功筛选出针对μPAmRNA靶序列的有效干扰序列，抑制效应持续至72h；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。

Inhibition of uPA Expression with siRNA in Mice Sertoli Cells

The aim of our study is to analyze the suppressive effects of siRNA targeting uPA gene （si- μPA） in uPA expression in mice sertoli cell line （TM4 cells）. 3 different sequences of si-μPA were transfected into TM4 cells to screen the effective si-μPA. 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-μPA, 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.