利用CRISPR/Cas9 RNP靶向表观遗传修饰技术调控成纤维细胞定向转分化为睾丸间质细胞及其分子机制研究

Leydig cells (LC), which have exclusive function of synthesizing and secreting androgens, are considered as the major source of androgen in mature males. It is well known that the lack of androgen results in congenital male genitals malformation, sexual dysfunction and sterility. In previous study, the mice fibroblasts were directly converted into functional Leydig-like cells by expressing three transcriptional factors related to LC development. However, it is notably that the reprogramming process is long, inefficient and generating induced Leydig-like cells with low-yielded androgen. Further analyses suggested that the stable expression of transcriptional factors and LC-specific genes is crucial for the reprogramming of fibroblasts into LC. We hypothesize that the reprogramming of fibroblasts into Leydig cells is mediated by the epigenetic alterations in the nucleus which drives the stable expression of endogenous steroidogenic genes. We propose to achieve the efficient reprogramming of skin fibroblasts into functional LCs using Cas9 ribonucleoprotein (RNP) complexes with targeting genome editing. Next we’ll identify the epigentic mechanism of cells reprogramming using DNA methylation analysis, chromatin immunoprecipitation assay, transcriptomics and bio-informatics methods. The research will provide the foundation of stem cell therapy for male hypogonadism.

睾酮主要由睾丸间质细胞(Leydig cell，LC)合成和分泌，其缺乏会导致男性性器官发育迟缓、性功能障碍及生育能力下降，严重影响男性的生殖健康和生活质量。课题组前期利用慢病毒载体过表达性腺发育相关的转录因子使成纤维细胞转分化为具雄激素合成功能的类LC，然而，分化时间长、效率低及细胞功能不稳定等缺点限制其在临床上的应用。进一步分析提示，内源LC谱系发育相关转录因子及调控基因的稳定表达是实现高效转分化的决定因素。本课题将围绕表观遗传修饰调控成纤维细胞定向转分化为LC这一科学问题，拟用具DNA甲基化编辑功能的Cas9核糖核蛋白复合物(RNP)靶向激活皮肤成纤维细胞中LC谱系关键基因的表达，使之重编程为LC并经动物模型验证其生物学功能。随后，利用转录组、ChIP及基因组甲基化分析等技术阐明表观遗传修饰调控成纤维细胞转分化的分子机制，为男性性腺机能低下综合征的细胞治疗提供科学依据和应用基础。

睾酮主要由睾丸间质细胞 (Leydig cell，LC)合成和分泌，其缺乏会导致男性生殖道发育异常、生育能力下降、性功能障碍、肌肉形成减少、骨质疏松症和认知功能障碍，严重影响男性的生殖健康和生活质量。移植具雄激素合成功能的类睾丸间质细胞（Leydig-like cell, LLC）是一种可治愈男性雄激素缺乏的有效疗法。但如何获得可满足临床需要的LLC仍是难点。本研究首先对 LC 和成纤维细胞中的全基因组 DNA 甲基化水平和表达谱进行联合分析，鉴定出潜在的、受表观遗传调控的信号通路及雄激素合成关键基因。然后，利用CRISPR/dCas9激活系统和信号通路调节因子组合，将雄性或雌性来源的成纤维细胞重编程为具LC特性的LLC。转录组分析结果显示，LLC和LC在全基因表达谱及转录因子表达模式方面，其Pearson相关性系数分别高于0.81和0.96，表明LLC在表型和基因型上与真正的LC非常接近。最后，在性腺功能减退模型动物的睾丸中移植LLC，可显著提升动物血清中及睾丸内的睾酮含量。在2型糖尿病模型大鼠腋窝皮肤移植LLC，同样可以提升模型动物血清睾酮水平，并同时改善其高血糖状态。本研究建立一种CRISPR/Cas9靶向表观遗传修饰技术，可以促使皮肤来源的成纤维细胞高保真度重编程为功能性LLC，为日后男性性腺功能减退症和代谢相关合并症的治疗提供临床级的细胞来源。