睾丸体细胞外泌体来源miR-204对睾丸器官重构作用机制研究

外泌体来源微小RNA和蛋白质参与机体组织细胞发育过程的调控，睾丸体细胞来源外泌体对睾丸器官形成和精子发生也发挥重要作用。这些外泌体所含蛋白质、脂质及核酸调控睾丸器官形成的具体机制尚不清楚。我们前期工作中，采用3D培养技术重构了睾丸生精小管；新近研究证实Dhh信号通路对睾丸重构有调控作用，利用生信技术发现若干个高表达的miRNA和蛋白质，高表达miR-204与DHH信号轴的GLI存在靶向结合位点。本项目拟在前期基础上，结合3D培养技术探究外泌体中与睾丸形成相关的miRNA或蛋白质的表达；体细胞外泌体来源miR204及其他miRNA和蛋白质对睾丸形成的作用；采用过表达、沉默技术、WB、qPCR等探究细胞来源外泌体miRNA和蛋白质在睾丸重构和精子发生的机制。项目将初步明确体细胞来源外泌体miR-204及其他miRNA/蛋白至的作用机制，为后续睾丸器官发育、精子发生、男性不育等研究提供理论依据。

Exsomal miRNAs and proteins play and important role in normal physiology and development of tissues and cells. Although mechanisms regulating testis cord formation and spermatogenesis have been well-studied, somatic-exosomes are not well characterized. How protein, lipid and nucleic acid from exosomes modulates the formation of testis and spermatogenesis. In our previous study, we have been achieved to reconstruct the seminiferous tubules using in 3-dimensional (D) re-aggregated culture of testicular cells. And we found that the formation of testis cord are regarded the regulation using cyclopamine, as DHH signaling inhibitors, disturbed the tubule-like structures; we identified several miRNAs，one is miR-204, which is expressed highly in the testis, showing targeted to GLI. Therefore, we aim to comprehensively study the role and mechanisms of regulating testis development underlying somatic-derived exosomes with 3-dimensional (D) and organ culture. We (1) identify the expression of miR-204 and unknown miRNA/protein from exosomal somatic in the testis; (2) explore the role of miR-204 and unknown miRNA/protein from exosomal somatic on testis cord formation through the behavior of Sertoli cells, germ cell and Leydig cell including proliferation, differentiation and polarization (3) investigate the mechanism of miR-204 and unknown miRNA/protein from exosomal somatic on testis cord formation the mRNA and protein expression of down-regulated of Dhh signaling or other pathways using over-expression, RNAi, Western blot and qPCR. We believe that the results derived from this study will provides a novel avenue and methods for further investigation of the improvement of testicular development, spermatogenesis and male infertility.