非基因整合建立人诱导多能性干细胞方法的优化研究

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（CBMNC）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CBMNC，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Episo—mal方法进行优化。结果表明：CBMNC采用红系培养液，培养8d，使用启动子为sFFV（spleen focus forming virus）的Episomal载体，在低氧（3％）条件下诱导，CBMNC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。

An Improved Method for Generating Integration-Free Human Induced Pluripotent Stem Cells

The genome instability and tumorigenicity of induced pluripotent stem ceils （iPSC） hinder their great potentials for clinical application. Using episomal vectors to generate iPSC is the best way to solve safety issues at present. This method is simple and the exogenous gene was not integrated into the host genome. However, the reprogramming efficiency for this method is very low and thus limits its usage. This study was purposed to improve episomal method for generating induced pluripotent stem cells from cord blood mononuclear cells （ CB MNC）, to establish integration-free iPSC technology system, and to lay the foundation for individualized iPSC for future clinical uses. To improve the reprogramming efficiency for iPSC, episomal method was used at various combinations of episomal vectors, pre - stimulating culture mediums and oxygen condition were tested to optimize the method. The results showed that using erythroid culture medium for culturing 8 days, transfecting with episomal vectors with SFFV （spleen focus forming virus ） promoter under the hypoxic condition （3%）, CB MNC could be mostly efficiently reprogrammed with the efficiency 0. 12%. Furthermore, the results showed that erythroblasts （CD36＋ CD71＋ CD235alow） were the cells that are reprogrammned with high efficiency after culture for 8 days. It is concluded that a highly efficient and safe method for generation of integration-free iPSC is successfully established, which is useable in clinical study.