用非整合重编程技术构建人外周血单个核细胞来源的诱导多能干细胞  

Generation of human induced pluripotent stem cells from peripheral blood mononuclear cells using the non-integrating reprogramming technique

作　　者：梁栋[1] 胡焕然[1] 李雅红[1] 刘安[1] 林颖[1] 马定远[1] 张菁菁[1] 王艳[1] 胡平[1] 许争峰[1] 

机构地区：[1]南京医科大学附属南京妇幼保健院产前诊断中心,南京210004

出　　处：《临床检验杂志》2015年第10期740-743,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81300495);江苏省临床医学重点项目(BL2012039);江苏省卫生厅科学项目(H201343)

摘　　要：目的用非整合重编程技术构建人外周血单个核细胞(PBMC)来源诱导多能干细胞(i PSC)。方法收集健康人外周血标本,分离PBMC,用含4个重编程转录因子(OCT3/4、SOX2、KLF4和c-MYC)的仙台病毒感染PBMC,并用无饲养层细胞的培养体系培育获得i PSC。通过细胞形态学、碱性磷酸酶(AP)染色、免疫荧光染色、拟胚体形成和分化实验对i PSC细胞进行多能性验证;用核型分析和仙台病毒基因组RNA检测对其进行安全性验证。结果在仙台病毒感染后16 d后,PBMC出现i PSC样克隆。i PSC可进行传代培养,且经多能性验证实验证明其具有多能性;安全性验证结果表明,i PSC核型正常,外源性病毒基因组RNA可被完全清除。结论建立了对PBMC进行非整合重编程的诱导体系,并获得了具有多能性、核型正常且不含外源基因的i PSC。Objective To construct the induced pluripotent stem cells（ i PSCs） from human peripheral blood mononuclear cells（ PBMC） using the non-integrating reprogramming technique. Methods Peripheral blood samples from healthy persons were collected and the PBMCs were isolated. Then,the PBMCs were infected with sendai virus containing four reprogramming factors— OCT3 /4,SOX2,KLF4 and c-MYC,and the h PBMC-derived i PSCs were obtained by the culture system with feeder-free condition. The multipotency of the obtained h PBMC-derived i PSCs was verified by the observation of cell morphology,alkaline phosphatase（ AP） staining,immunofluorescence staining,and the embryoid body formation and differentiation assay,and its safety was evaluated by the karyotype assay and the detection of sendai virus genomic RNA. Results i PSC-like colonies emerged at day 16 after the PBMCs were infected with sendai virus. The obtained i PSCs were able to be cultured with a few passages,and were demonstrated to have the multipotency. Moreover,the h PBMC-derived i PSCs carried normal karyotype,and no any exogenous genomic RNA from sendai virus was detected in it.Conclusion A non-integrating reprogramming system for h PBMC was successfully constructed,and the i PSCs from h PBMC with multipotency,normal karyotype and the lack of exogenous genes were obtained.

关 键 词：人外周血单个核细胞 重编程 诱导多能干细胞 非整合 仙台病毒 

分 类 号：R394.2[医药卫生—医学遗传学]