构建阵挛性癫痫伴破碎红纤维综合征患者来源的诱导多能干细胞系  

Generation of induced pluripotent stem cell lines from a MERRF patient

作　　者：胡焕然[1] 徐天慧[1] 梁栋[1] 罗春玉[1] 林颖[1] 季修庆[1] 胡平[1] 许争峰[1] 

机构地区：[1]南京医科大学附属妇产医院产前诊断中心,南京210004

出　　处：《临床检验杂志》2016年第8期584-589,共6页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81300495);江苏省妇幼保健科研课题基金(F201314);江苏省卫生厅医学科研立项课题(H201343);江苏省科技厅社会发展项目(BE2015614)

摘　　要：目的构建阵挛性癫痫伴破碎红纤维综合征(MERRF)患者来源的诱导多能干细胞(i PSCs)。方法采集MERRF患者外周血并分离PBMC,用含有4个重编程转录因子(OCT4、SOX2、KLF4和c-MYC)的仙台病毒感染PBMC,获得患者来源的i PSCs;用细胞形态学、免疫荧光染色、拟胚体形成分化和RT-PCR对i PSCs的多能性进行鉴定;核型分析验证其安全性;焦磷酸测序检测患者来源的i PSCs中线粒体DNA(mt DNA)第8344位的突变比例。结果患者来源的i PSCs经过多能性验证实验证明其具有多能性;核型分析表明,i PSCs核型正常;建立的6个i PSCs细胞系中,mt DNA 8344位点的突变比例分别为68.24%、60.51%、45.95%、24.06%、62.11%和0。结论通过非整合重编程技术成功构建MERRF患者来源i PSCs细胞系,为进一步研究MERRF疾病提供多能干细胞资源。Objective To generate induced pluripotent stem cell （iPSCs） from myoclonic epilepsy and ragged-red fibers （MERRF） patient. Methods The peripheral blood sample from a MERRF patient was collected and peripheral blood mononuclear cells （PBMC） were isolated. The PBMC were infected with Sendai virus containing four reprogramming transcription factors-OCT4, SOX2, KLF4 and c-MYC. The pluripotency of the obtained iPSCs from the patient was verified by eel1 morphology, immunofluorescence staining, forma- tion and differentiation assay of the embryoid body （EB） and RT-PCR. Karyotypes of the iPSCs were revealed by GTG-banding assay and the safety of the iPSCs was verified by karyotype analysis. The ratios of mitochondrial carrying m. 8344 point mutation were detected by pyrosequencing. Results The MERRF patient-derived iPSCs were demonstrated to possess the pluripotency, and their karyotypes were normal. The ratios of mitoehondrial carrying m. 8344 mutant were 68.24%, 60.51%, 45.95%, 24.06% , 62.11% and 0 respectively in 6 generated iPSCs lines. Conclusion We generated iPSCs lines derived from a MERRF patient by non-integrating reprogramming method. These iPSCs may provide model for the further investigation on MERRF.

关 键 词：诱导多能干细胞 重编程 阵挛性癫痫伴破碎红纤维综合征 仙台病毒 异质性 

分 类 号：R394.2[医药卫生—医学遗传学]