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Urokinase Receptor Gene Expression Inhibited by siRNA cocktails in co-culture of Spermatogenic cells with Sertoli cells from 3-Week-Old Rat

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Journal of Reproduction and contraception 2010年第2期21卷 67-77页

作者： Dong-hui HUANG Hu ZHAO Yu MING Cheng-liang XIONG Family Planning Research Institute Tongji Medical College Huazhong University of Science & Technology Wuhan 430030 China Department of Human Anatomy Tongji Medical College Huazhong University of Science & Technology Wuhan 430030 China

Objective To study the functions of urokinase receptor （uPAR） during spermatogenesis by adding targeted siRNA cocktails to the co-culture of Sertoli cell with spermatogenic cells. Methods The seminiferous tubule samples from SD rats aged 20-23 d were digested with collagenase for 15-20 rain, then were cut into smaU fragments. Tubular fragments were digested with collagenase again for 5 -10 min, then gently resuspended in F12/ DMEM supplemented with vitamins and hormones. cell samples were seeded in plate and cultured at 32 ℃ for 2 weeks. After 48 h, siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were introduced into this cocultured Sertoli/spermatogenic cells. After then for 48 h, expression of uPAR mRNA was analyzed by real-time RT-PCR. Results Some spermatogenic cells took place morphologic changes and generated an obvious flagellum. The expression levels of uPAR mRNA silencing with siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were significantly lower than those in nontransfected group （P〈0.05）; the inhibition rate was 63.5% or 76.7%, respectively. conclusion The meiosis and spermiogenesis could take place in this culture system, and the siRNA cocktails can effectively inhibit the gene expression of uPAR in co-cultured Sertoli/spermatogenic cells.

关键词： small interfering RNA RNA interference urokinase receptor (uPAR) spermatogenic cell cell co-cultures

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