CAJ | 학술논문

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.
목적 연구휴대유인Ⅰ형납리자통도기인α아기(SCN1A)기인c.5768A＞G잡합돌변적유도다능성간세포(iPSCs)재라서체내적다향분화능력. 방법 장인원SCN1A c.5768A＞G잡합돌변iPSCs세포주분별주사도3지NOD/SCID소서동측전지피하조직화후지기육조직중,40 d후관찰성류정황,기록기태류대소;취출기태류조직제작석사절편,진행HE염색,관찰기태류적세포형태특정;제취기태류조직기인조DNA,PCR확증대유c.5768A＞G돌변위점적편단병대기진행측서급생물신식학분석. 결과 주사iPSCs 40 d후,1지NOD/SCID소서전지피하화후지기육중균유기태류형성;HE염색현시기태류조직중지소유3충류형적세포,분별위원우내배층적선체세포、원우중배층적지방세포화원우외배층적성섬유세포;측서분석현시기태류조직기인조중존재SCN1A기인c.5768A＞G잡합돌변;생물신식학분석현시해위점위고위험성돌변. 결론 인원SCN1A기인c.5768A＞G잡합돌변형iPSCs구유분화성삼배층적다향분화능력.
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.