Wang Hong, Yuan Qingqing, Niu Minghui, Wen Liping, Fu Hongyong, Zhou Fan, Zhang Weihui, He Zuping
State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Shanghai Institute of Andrology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Methods Mol Biol. 2018;1748:191-202. doi: 10.1007/978-1-4939-7698-0_14.
Sertoli cells, as the unique somatic cells within the seminiferous tubules, play essential roles in regulating normal spermatogenesis. In addition, recent studies have demonstrated that Sertoli cells could have significant applications in regenerative medicine due to their great plasticity. However, the roles of genes in controlling the fate determinations of human Sertoli cells remain largely unknown. Silencing genes of human Sertoli cells utilizing small interfering RNAs (siRNAs) is an important method to explore their functions and mechanisms in human Sertoli cells. We isolated and identified human Sertoli cells. RNA interference (RNAi) was employed to probe the roles and signaling pathways of BMP6 and BMP4 in mediating the proliferation and apoptosis of human Sertoli cells. Specifically, siRNAs against BMP6 and BMP4 were used to knock down the expression levels of BMP6 and BMP4 and examine the function and mechanism in controlling the fate decisions of human Sertoli cells. In this chapter, we provided the detailed methods of RNAi in silencing BMP6 gene of human Sertoli cells. Quantitative real-time PCR demonstrated that the designed BMP6 siRNAs apparently silenced BMP6 mRNA in human Sertoli cells at 24 h after transfection. Western blots showed that the siRNAs silenced the expression of BMP6 protein effectively at 48 h after transfection. In summary, siRNAs can effectively and specifically knock down targeting genes at both transcriptional and translational levels utilizing RNAi in human Sertoli cells.
支持细胞作为生精小管内唯一的体细胞，在调节正常精子发生过程中发挥着重要作用。此外，最近的研究表明，支持细胞因其高度可塑性在再生医学中具有重要应用前景。然而，基因在控制人支持细胞命运决定中的作用仍 largely未知。利用小干扰RNA（siRNA）沉默人支持细胞基因是探索其在人支持细胞中功能和机制的重要方法。我们分离并鉴定了人支持细胞。采用RNA干扰（RNAi）技术探究骨形态发生蛋白6（BMP6）和骨形态发生蛋白4（BMP4）在介导人支持细胞增殖和凋亡中的作用及信号通路。具体而言，使用针对BMP6和BMP4的siRNA敲低BMP6和BMP4的表达水平，并研究其在控制人支持细胞命运决定中的功能和机制。在本章中，我们提供了RNAi沉默人支持细胞BMP6基因的详细方法。定量实时PCR结果显示，设计的BMP6 siRNA在转染后24小时能显著沉默人支持细胞中的BMP6 mRNA。蛋白质印迹分析表明，siRNA在转染后48小时能有效沉默BMP6蛋白的表达。总之，利用RNAi技术，siRNA可在转录和翻译水平有效且特异性地敲低人支持细胞中的靶向基因。
