多发性内分泌腺瘤病1型患者血液细胞来源特异性诱导多能干细胞的建立

Generation of multiple endocrine neoplasia 1 patient-specific induced pluripotent stem cells from blood cell

目的:建立多发性内分泌腺瘤病1型(multiple endocrine neoplasia 1,MEN1)患者血液细胞来源的特异性诱导多能干细胞(induced pluripotent stem cells,iPSCs)模型。方法:将表达Oct4、Sox2、Lin28、L-Myc和Klf4转录因子的oriP/EBNA1附着体电转染MEN1患者血液细胞使其重编程获得iPSCs,并通过碱性磷酸酶染色、核型鉴定、基因测序、RT-PCR反应、畸胎瘤形成实验及类胚体形成实验检测其特性。结果:获得的i PSCs碱性磷酸酶染色呈阳性,核型正常,测序结果显示在其第9号外显子存在c.1288 G>T位点突变,表达干细胞多能性基因Sox2、Oct4、Nanog和Klf4和Lin28,体内畸胎瘤形成实验可分化为内、中、外三胚层细胞,体外可形成类胚体。结论:成功获得携带第9号外显子c.1288 G>T位点突变的MEN1患者血液细胞来源的特异性iPSCs,为后续机制研究奠定基础。

Objective：To establish patient - specific induced pluripotent stem cells （ iPSCs ） model of multiple en-docrine neoplasia 1（MEN1） patients blood cells. Methods： Blood cells from MEN1 patient were infected by episomal vectors with an oriP/EBNA - 1 （ Epstein - Barr nuclear antigen - 1） backbone for delivering the reprogramming genes Oct4, Sox2, Lin28 ,L - Myc,and Klf4 to generate iPSCs. The charateristic of the obtained iPSCs were verified by alka-line phosphatase staining,karyotyping analysis,gene sequencing,RT - PCR,teratoma test,and the formation of embry- oid body. Results ： Alkaline phosphatase staining were positive. The obtained iPSCs carried normal karyotype and ex-pressed the pluripotent genes Sox2, Oct4, Nanog, Klf4 and Lin28. Sequencing analysis showed that it had a heterozy-gous G 〉 T mutation on the exon 9 of Menl gene. In the teratoma test, three germ layer cells were found. EBs were formed by suspended culture. Conclusion：The iPSCs from MEN1 patien ts blood cells with multipotency could be suc-cessfully generated, which lay the foundation of further mechanism study.