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诱导多能性干细胞(iPSC)技术具有临床应用前景,但iPSC的遗传稳定性和成瘤性阻碍了其可能的临床应用。非整合质粒(Episomal)方法无外源基因整合到宿主基因组上并且方法简单,适宜推广,是目前保证iPSC遗传安全性的最佳方案之一,但其诱导效率偏低,严重阻碍了其应用。本研究旨在优化Episomal方法,将脐血单个核细胞(CB MNC)重编程为诱导多能性干细胞(iPSC),建立无基因整合的iPSC的高效生成技术体系,为以后建立疾病iPSC奠定基础。利用CB MNC,通过比较不同氧含量,诱导质粒,MNC培养方法和预刺激时间等条件对Episomal方法进行优化。结果表明:CB MNC采用红系培养液,培养8 d,使用启动子为SFFV(spleen focus forming virus)的Episomal载体,在低氧(3%)条件下诱导,CB MNC重编程效率最高,可达到0.12%。通过分析最佳条件下供体细胞成分发现,表型为CD36+CD71+CD235alow的有核红细胞是重编程最主要的供体细胞来源。结论:本研究成功建立并优化出一种可推广的高效安全的,可以用于临床应用研究的iPSC诱导技术。
Induced pluripotent stem cells (iPSC) technology has clinical application prospects, but the genetic stability and tumorigenicity of iPSC hinder its possible clinical application. Episomal method is one of the best protocols to ensure the genetic safety of iPSC without foreign gene integration into the host genome. The method is simple and suitable for generalization, but its low induction efficiency seriously hinders its application. This study aimed to optimize the Episomal method, to reprogram cord blood mononuclear cells (CB MNCs) to induce pluripotent stem cells (iPSC), and to establish an efficient generation system for genetically-integrated iPSCs to lay the foundation for future establishment of iPSCs. Using CB MNC, the Episomal method was optimized by comparing different oxygen content, plasmid induction, MNC culture and pre-stimulation time. The results showed that CB MNC was cultured in erythroid medium for 8 days and induced by hypoxia (3%) using Episomal vector with promoter SFFV (spleen focus forming virus). The CB MNC reprogramming efficiency was the highest 0.12%. By analyzing the donor cell composition under the optimal conditions, it was found that the nucleated erythrocytes with the phenotype of CD36 + CD71 + CD235alow were the most important donor cells for reprogramming. Conclusion: This study successfully established and optimized a kind of iPSC induction technology that can be popularized and safe and can be used in clinical research.