人类体细胞核移植胚染色体不稳定的机制研究

Human somatic cell nuclear transfer(SCNT) is one of the major methods to derive patient-tailored pluripotent stem cells and to understand reprogramming. But till now, breakthough is still needed for human SCNT technique, for the low blastocyst development rate and unsuccessful to derive pluripotent stem cells. A widely accepted notion is that imcomplete reprogramming accounts for development failure. We have recently done single cell CGH analysis on developmentally arrested human SCNT embryos. The results suggest that their chromosome duplication and deletion are more severe than that of nomal IVF developmentally arrested ones, most of the SCNT embryos display chaotic phenomenon , which indicates that abnormal chomosome segregation might be another important reason for developmental arrest. In this project, we plan to conduct SCNT with donor cells carrying stably integrated Nanog-GFP and H2B-RFP, monitoring and recording the relationship between chromosome behavior and reprogramming process through confocal fluoresence microscope. Combined with genome-wide chomosome analysis by whole comparative genomic hybridization in arrested reconstitute embryos, and the expression analysis of SAC related proteins in developmentally arrested human SCNT embryo and blastomeres and immunohistochemistry and gene expression analysis of SAC in the single cell lever. It can help us understand the relationship and machanism between reprogramming and abnormal mitosis. Based on it, the influence of potential interventions to the SCNT effieciency will be evaluated.

人类体细胞核移植（SCNT）是构建患者特异性多能干细胞和研究重编程机制的主要方法之一，目前该技术仍有待突破。普遍观点认为SCNT效率低与重编程不完全有关。本项目组近期对人SCNT发育阻滞胚进行单细胞CGH分析，发现其相对体外受精阻滞胚存在更严重的染色体重复及缺失，大多数出现染色体混乱现象，提示不正常染色体分离可能也是影响重构胚发育的重要因素。本研究拟在此工作基础上，通过构建稳定整合H2B-RFP和Nanog启动子-GFP的患者成纤维细胞做为供核细胞用于SCNT研究，对比去核和不去核SCNT方法，利用实时成像技术动态观察重构胚早期发育过程中染色体分离行为和重编程进程的关系，并对发育阻滞胚和分裂中期卵裂球开展单细胞水平上的全染色体组分析，纺锤体组装检控点相关蛋白的免疫组化分析和基因表达分析，以全面了解重编程和异常有丝分裂之间的关系和潜在的分子机制，探讨可能干预措施对SCNT效率的影响。

人类体细胞核移植（SCNT）是构建患者特异性多能干细胞和研究重编程机制的主要方法，但人类SCNT效率很低。本项目对人类去核SCNT发育阻滞胚进行单细胞CGH分析，发现其存在严重的染色体重复及缺失，大多数呈现染色体混乱现象，提示不正常染色体分离可能是影响重构胚发育的重要因素。不去核SCNT胚胎的囊胚形成率显著高于去核SCNT胚胎（22.7% vs 2.0%），不去核SCNT囊胚已成功建立胚胎干细胞系。另一方面，将精子核注入去核SCNT重构胚后，重构胚的囊胚形成率达到30%，并成功建立胚胎干细胞系，说明精子核也能帮助提高重构胚的发育潜能。我们还探索了人类孤雄生殖的方法，并建立了首株人类孤雄来源的干细胞系。