Ⅰ型纳离子通道基因α亚基c.5768A＞G突变型多能性干细胞多向分化能力的研究  

Pluripotency of induced pluripotent stem cells originated from an epileptic patient with sodium channel α-subunit type 1 c.5768A〉G mutation

作　　者：李文斌[1] 华立栋 张林明[1] 王超[1] 汤斌[1] 秦兵[2] 廖卫平[1] 石奕武[1] 

机构地区：[1]广州医科大学附属第二医院神经致病基因与离子通道病省部共建教育部重点实验室,广州510260 [2]广东省人民医院神经内科、广东省医学科学研究院,广州510060

出　　处：《中华神经医学杂志》2014年第5期463-466,共4页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81071045）;广州市科信局项目（2013J4100044）;中国科学院广州生物医药与健康研究院开放课题（KLRB201213）

摘　　要：目的 研究携带有人Ⅰ型纳离子通道基因α亚基（SCN1A）基因c.5768A＞G杂合突变的诱导多能性干细胞（iPSCs）在裸鼠体内的多向分化能力. 方法 将人源SCN1A c.5768A＞G杂合突变iPSCs细胞株分别注射到3只NOD/SCID小鼠同侧前肢皮下组织和后肢肌肉组织中,40 d后观察成瘤情况,记录畸胎瘤大小;取出畸胎瘤组织制作石蜡切片,进行HE染色,观察畸胎瘤的细胞形态特征;提取畸胎瘤组织基因组DNA,PCR扩增带有c.5768A＞G突变位点的片段并对其进行测序及生物信息学分析. 结果 注射iPSCs 40 d后,1只NOD/SCID小鼠前肢皮下和后肢肌肉中均有畸胎瘤形成;HE染色显示畸胎瘤组织中至少有3种类型的细胞,分别为源于内胚层的腺体细胞、源于中胚层的脂肪细胞和源于外胚层的成纤维细胞;测序分析显示畸胎瘤组织基因组中存在SCN1A基因c.5768A＞G杂合突变;生物信息学分析显示该位点为高危险性突变. 结论 人源SCN1A基因c.5768A＞G杂合突变型iPSCs具有分化成三胚层的多向分化能力.Objective To explore the pluripotency of induced pluripotent stem cells （iPSCs） originated from an epileptic patient with sodium channel α-subunit type 1 （SCN1A） c.5768A〉G mutation.Methods The iPSCs with human SCN1A c.5768A〉G mutation were injected into the hypodermis and muscle in forelimb and hindlimb of three NOD/SCID mice for teratoma forming.The teratomas size was observed and measured 40 days after the injection and the blastoderm cell morphology of the teratoma was identified by using Hematoxylin-Eosin staining.Genomic DNA of the teratoma tissues was got and PCR amplification was performed on the fragments carried c.5768A〉G mutation; and sequencing analysis was performed by direct sequencing and bioinformatics.Results Teratomas was formed in the hypodermis and muscle of a NOD/SCID mouse 40 days after injection.Three tissues differentiated from endoderm,mesoderm and ectoderm of the teratomas were observed.Sequence analysis showed that the teratoma cells carried c.5768A〉G mutation and the mutation site was highly conserved in different species,which had a probably damage to SCN1A protein.Conclusions The iPSCs originated from human SCN1A c.5768A〉G mutation have multiple potency to differentiate into endoderm,mesoderm and ectoderm.

关 键 词：离子通道 突变 诱导多能性干细胞 多向分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]