摘要目的 探讨体外定向诱导小鼠诱导性多潜能干细胞(induced pluripotent stem cell,iPSC)向耳蜗毛细胞及螺旋神经节细胞分化的可行性.方法 建立小鼠iPSC培养体系并对iPSC加以鉴定.分离出生3d小鼠的耳蜗Corti器,将小鼠iPSC与耳蜗Corti器共培养.对产生的分化细胞用毛细胞标志物肌球蛋白Ⅶa(Myosin Ⅶa),Math1(碱性螺旋-环-螺旋转录因子家族成员),钙视网膜蛋白(Calretinin),Espin(F肌动蛋白的结合蛋白)进行免疫细胞化学鉴定.分离出生3d小鼠的耳蜗蜗轴,将小鼠iPSC与耳蜗蜗轴共培养,对分化细胞用神经元的标志物神经上皮干细胞蛋白(Nestin)、中等分子量神经丝蛋白(Neurofilament-M)、β-Ⅲ微管蛋白(β-ⅢTubulin),Ⅰ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 1,VGluT1)进行免疫细胞化学鉴定.结果 免疫细胞化学染色结果显示,小鼠iPSC与耳蜗Corti器共培养18 d后可得到表达毛细胞标志蛋白MyosinⅦa、Math1、Calretinin 及Espin的细胞;小鼠iPSC与耳蜗蜗轴共培养18 d后可得到表达神经元标志蛋白Nestin、Neurofilament-M、β-ⅢTubulin,VGluT1的细胞.结论 共培养方法可成功将小鼠iPSC诱导分化为表达毛细胞及螺旋神经节细胞分子标志物的细胞.

abstractsObjective In this study,we investigated the potential of mouse induced pluripotent stem cells(iPSC) for use as a source of transplants for the restoration of auditory hair cells and spiral ganglion neurons.Methods We co-cultured the mouse iPSC with the cells of the cochlear organ of Corti or the modiolus in vitro.The cochlear organ of Corti (which contains cochlear hair cells) and the modiolus (which contains auditory spiral ganglion neurons) were obtained from postnatal day 3 (P3) CD-1 ICR mice.After 18 days of coculture with the cells of newborn mouse cochleae.The expressions of hair cell markers (Myosin Ⅶ a,Math1,Calretinin,Espin) and Spiral ganglion neuron markers [Nestin,Neurofilament-M,β-Ⅲ Tubulin,Vesicular glutamate transporter 1 (VGluT1)] were detected by immunocytochemical analysis.Results Immunocytochemical analysis results indicated that the differentiated iPSC expressed auditory hair cell markers (Myosin Ⅶ a,Math1,Calretinin,Espin) and spiral ganglion markers (Nestin,Neurofilament-M,β-Ⅲ Tubulin,VGluT1).Conclusion Mouse iPSC in virto cultured could successfully be induced to differentiate into hair cell-like cells and spiral ganglion-like cells with hair cell and spiral ganglion molecular markers.