构建脆性X染色体综合征患者来源的诱导多能干细胞系  

Construction of induced pluripotent stem cell lines from patients with fragile X syndrome

作　　者：包勤敏 梁栋[1] 乔凤昌 王艳[1] 马定远[1] 张翠平 胡平[1] 许争峰[1] BAO Qinmin;LIANG Dong;QIAO Fengchang;WANG Yan;MA Dingyuan;ZHANG Cuiping;HU Ping;XU Zhengfeng(Department of Prenatal Diagnosis,Women′s Hospital of Nanjing Medical University,Nanjing Maternity and Child Health Care Hospital,Nanjing 210004,Jiangsu,China)

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》2020年第3期164-170,共7页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81300495)。

摘　　要：目的构建脆性X综合征(FXS)患者来源的诱导多能干细胞(iPSCs)。方法采集2个FXS家系患者外周血并分离外周血单个核细胞(PBMC),用含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iPSCs;采用细胞形态学、免疫荧光染色等技术对iPSCs进行多能性鉴定;核型分析验证其基因组完整性;利用PCR扩增结合微流控毛细管电泳技术检测重编程前后CGG重复数。结果成功对2个脆性X家系患者的PBMC细胞进行了重编程。患者来源的iPSCs经过多能性验证试验证实,其表达多能性标志基因并具有向三胚层分化潜能;核型分析结果证实iPSCs核型正常;2例先证者细胞系重编程前后的CGG重复数差异无统计学意义。结论通过非整合重编程技术成功构建2个FXS患者来源iPSCs细胞系,为进一步研究FXS疾病提供多能干细胞资源。Objective To construct the induced pluripotent stem cell lines(iPSCs)from fragile X syndrome(FXS)patients.Methods The peripheral blood samples from two FXS patients were collected,and peripheral blood mononuclear cells(PBMCs)were isolated.These PBMCs were transfected with Sendai virus containing four reprogramming transcription factors,including OCT4,SOX2,KLF4 and c-MYC,so as to obtain the iPSCs from FXS patients.The pluripotency of the obtained iPSCs was identified by cell morphology and immunofluorescence staining.The genome integrity of the iPSCs was verified by karyotype analysis.The CGG repeats in the genome before and after reprogramming were determined by PCR amplification combined with microfluidic capillary electrophoresis.Results The PBMCs from two FXS patients were reprogrammed successfully.The obtained iPSCs were demonstrated to possess pluripotency,express the pluripotency marker genes,and have the potential to differentiate into the three germ layers.The results of karyotype analysis showed that iPSCs were normal in karyotype.There was no significant difference in the number of CGG repeats before and after reprogramming.Conclusion The iPSCs from two FXS patients are constructed successfully by the non-integrating reprogramming technology,which provide pluripotent stem cells for further studying FXS.

关 键 词：诱导多能干细胞 重编程 脆性X综合征 仙台病毒 CGG重复数 

分 类 号：R394.2[医药卫生—医学遗传学]