CLCN2基因突变者尿液来源诱导多能干细胞的构建及定向分化神经细胞  

Establishment of urine-derived induced pluripotent stem cell line from one patient carrying homozygous mutations in CLCN2 gene and differentiation into neural cells

作　　者：陈灼林 刘佳[1] 谢冰冰[2] 甘周庆 钟秀风[2] 彭福华[1] Chen Zhuolin;Liu Jia;Xie Bingbing;Gan Zhouqing;Zhong Xiufeng;Peng Fuhua(Department of Neurology,the Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)

机构地区：[1]中山大学附属第三医院神经内科,广州510630 [2]中山大学中山眼科中心国家重点实验室,广州510060

出　　处：《新医学》2020年第8期581-589,共9页Journal of New Medicine

基　　金：广东省精准医学与干细胞重大专项(2017B020230003)。

摘　　要：目的建立和鉴定CLCN2基因突变患者尿液来源特异诱导多能干细胞(iPSC),并初步探讨其神经分化能力。方法收集临床上1例CLCN2基因突变患者尿液细胞,将表达重编程因子OCT4、SOX2、KLF4和SV40LT的附加型载体通过电转染导入尿液细胞,使其重编程为iPSC,并继续诱导其神经分化。通过Sanger测序、核型鉴定、免疫荧光、逆转录PCR、畸胎瘤实验及定向分化等评价干细胞和神经细胞特性。结果电转后部分尿液细胞发生形态改变,形成边界清楚的致密细胞克隆团;这些克隆状生长的细胞可以连续传代,具有正常核型,含有CLCN2基因无义突变,表达多能干性标记且无外源基因;裸鼠体内形成三胚层畸胎瘤;体外可定向诱导分化为神经干细胞和星形胶质细胞。结论CLCN2基因突变患者尿液细胞可重编程为iPSC,并可体外分化为神经细胞,可为CLCN2基因突变相关疾病的研究提供重要材料。Objective To construct and identify the urine-derived induced pluripotent stem cells(iPSC) from one patient carrying homozygous mutations in CLCN2 gene,and to initially explore their ability of neural differentiation.Methods Urine cells from one patient with CLCN2 gene mutation were collected and transfected with episomal plasmids carrying the OCT4,SOX2,KLF4 and SV40 LT to be reprogrammed into iPSC,and then be differentiated into neural cells.The characteristic of the iPSC and the neural cells were verified by Sanger sequencing,karyotyping analysis,immunocytochemistry,RT-PCR,teratoma test,directed differentiation and so on.Results The transfected urine cells gradually appeared some clonal cell clusters with clear boundary,which could be continuously passaged,showed normal karyotype,contained homozygous CLCN2 mutation and expressed pluripotent markers without integration of exogenous genes.Three germ-layer teratomas were identified in the nude mice.These iPSC could be induced to differentiate into neural stem cells and astrocytes.Conclusion Urine cells from one patient carrying homozygous mutations in CLCN2 gene can be reprogrammed into iPSC and neural cells,which can provide vital evidence for investigating CLCN2 gene mutation-associated diseases.

关 键 词：诱导多能干细胞 CLCN2 白质脑病 神经分化 

分 类 号：R741[医药卫生—神经病学与精神病学]