泛素激活酶E1同系物UBA1对核移植胚胎干细胞来源的雄性生殖细胞分化及功能的影响

The incidence of male infertility is increasing. Advanced techniques like Stem cell culture generate differentiated germ cells, but these germ cells were immotile or showed reduced motility. Ubiquitin-proteasome pathway is important for cellular protein turnover and protein homeostasis. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. It regulates many basic cellular processes, such as antigen presentation, signal transduction and cell cycle. And UBA1 plays an important role in sperm physiological processes such as capacitation and motility. Along this line of inquiry, we utilized the NTESCs line as a model to study the function of UBA1 as follows: (1) The open reading frame of UBA1 is obtained by RT-PCR and inserted into a lentiviral vector and siRNA lentiviral silencing vectors. Utilized two-dimensional gel electrophoresis to identify a serial of proteins with altered expression. Comparison of the protein expression level relative to UBA1. (2) Established NTESCs differented to the male germ cell models in vitro. Through the overexpression and silenceexpression of UBA1 gene to observe the process of germ cell differented from nuclear transfer embryonic stem cells,and investigate the possible mechanisms.(3) Established a stable recipient mouse dyszoospermia model. Transplanted differented NTESCs into recipient mouse testis. Analyze the expression of UBA1 and its related proteins of potential functional relevance on spermatozoa. Through the above research to explain the ture role of UBA1 played on the process of NTESCs dieffrented to the male germ cell, Further improve the conditions of differentiation, and lay the foundation for the clinical carried out NTESCs transplantation in the treatment of male infertility.

男性不育症发病率逐年提高，其中内源性生殖细胞缺如所致无精子症或精子畸形综合征无法得到有效治疗。目前干细胞疗法已可生产精子，但有其局限性及不足，其分化条件及机制尚不清楚。已证实UBA1表达与雄性生殖细胞功能密切相关，本研究在体细胞核移植生产NTESCs技术平台的基础上：（1）构建含UBA1过表达及沉默表达重组慢病毒载体，感染NTESCs使用蛋白质组双向电泳筛选与UBA1相互作用的蛋白并分析；（2）建立NTESCs向雄性生殖细胞分化体外模型，观察UBA1对干细胞向雄性生殖细胞转化过程的影响并探讨作用机制；（3）将上述诱导分化得到的雄性生殖细胞注射到生精缺陷小鼠模型的睾丸，观察UBA1及其相关蛋白的表达与潜在精子功能的相关性。通过上述研究明确NTESCs向雄性生殖细胞分化过程UBA1所起的作用及机制，探索完善定向诱导分化的条件，为NTESCs移植治疗男性不育症提供理论依据。

体细胞核移植技术与胚胎干细胞技术结合产生的NT-ESCs，不仅可以用于基础研究，也能用于组织修复和再生医学。多项研究已提示，由ESCs获得精子的技术是可行的, 并且已证明体外诱导ESCs获得的精子能够受精并产生后代。所以，NT-ESCs向男性生殖细胞分化有可能同时解决移植物来源和免疫排斥这两大难题。本研究构建成功的小鼠NT-ESCs从形态特征、生长特性、染色体结构、多能性和全能性等各方面均符合小鼠胚胎干细胞的一系列特性并具有多能性，这为进一步研究组织修复及再生提供了技术平台。. 本课题通过模拟体内分化所需要的早期胚胎环境形成NTEBs，经添加维甲酸诱导增殖来优化胚胎干细胞的诱导分化情况，共培养诱导分化后的细胞表现出雄性生殖细胞的相关蛋白。 通过构建包含UBA1基因的慢病毒表达载体：GV358-UBA1,沉默表达载体GV248-UBA1 RNAi；经过细胞样本TMT蛋白数据分析筛选出与UBA1相关的差异蛋白Ctbp1,Psen2,Hes1，Dtx1,Dtx4,Trp53,Wnt2b,Racl,Rhoa；在研究过程中课题组发现UBA1过表达可以促进NTESCs诱导分化为雄性生殖细胞的进程；同时观察到细胞周期蛋白Cyclins A，B1，E3，D，细胞周期蛋白依赖性激酶CDK1，CDK2在体外定向诱导分化过程中的表达情况；初步揭示了UBA1可能通过对Notch和Wnt途径的调控来参与雄性生殖细胞的体外分化过程，对于进一步研究NT-ESCs的诱导分化打下基础。