先天性马蹄内翻足诱导多能干细胞建立及鉴定

Establishment and identification of congenital clubfoot induced pluripotent stem cells

目的 以尿液脱落细胞为来源,构建先天性马蹄内翻足患者特异的诱导多能干细胞（iPSCs）.方法 收集先天性马蹄内翻足患者尿液180 ml,通过离心,分离出尿液脱落细胞并进行培养,然后使用病毒系统对该细胞进行重编程,获得状态良好的细胞并进行鉴定,鉴定方法包括多能性基因检测、碱性磷酸酶（ALP）染色、核型鉴定、流式细胞仪（FACS）检测表面标志物、拟胚体（EB）悬浮球实验和甲基化检测等多种检测手段.结果 获得的细胞多能性基因表达与人胚胎干细胞H9相似,ALP染色呈强阳性,核型正常,多能性标记Tra-1-60阳性率为84.1％,能形成大量EB悬浮球,细胞在Nanog同源框（NANOG）和有机阳离子/肉毒碱运输蛋白4（Oct4）基因启动子区域的甲基化基本消失,证实获得的细胞具有多能性干细胞的所有特征,确定为iPSCs.结论 本实验成功构建了先天性马蹄内翻足患者特异iPSCs,为明晰先天性马蹄内翻足的演变特征提供了疾病模型.

Objective To establish specific induced pluripotent stem cells （iPSCs） fromthe patient with congenitalclubfoot by taking exfoliated cells in urine as source.Methods 180 ml of urine from clubfoot patient were collected,and the exfoliated cells in urine were isolated and cultured by centrifugation.Then,the cells were reprogrammed with virus system and identified by pluripotency gene detection,alkaline phosphatase （ALP） staining,karyotype identification,Fluorescein activated cell sorter （FACS） surface markers detection,embryoid body （EB） suspension ball assay and methylation assay.Results The expression of pluripotentgenes between obtained cellsand human embryonic stem cells H9 was similar,ALP staining showed strong positive.The cells possessed normal karyotype and 84.1% positive rate of pluripotency marker Tra-1-60,they could form a large number of EB suspension balls and almost lost methylation of the promoter regionsin Nanog homeobox （NANOG） and organic cation/carnitine transporter4 （Oct4） genes.Therefore,werevealed that the obtained cells owned all of the characteristics of pluripotent cells,and wecan identified them as iPSCs.Conclusion iPSCs were successfully constructed in this study,which provided cell materials for the clarification of evolution characteristics of congenital clubfootdisease.