新生大鼠耳蜗前体细胞的培养及鉴定

Culture and identification of cochlear progenitor cells from newborn rats

目的对新生大鼠耳蜗前体细胞进行分离、培养和鉴定。方法从出生7天的大鼠耳蜗中分离、培养前体细胞,用免疫细胞化学的方法对前体细胞进行鉴定;血清诱导分化后鉴定分化潜能,进一步了解其多向分化特性。结果原代培养的细胞,培养1d后即可见"细胞球"。细胞球内大部分细胞呈nestin、musashi1、pax2和BrdU阳性,表明其具有自我更新及有丝分裂的能力。细胞球经诱导分化14d后,对分化细胞行免疫细胞化学鉴定,发现分化细胞表达毛细胞标志物myosin VIIA和phalloidin,表达成熟神经元标志物NeuN,表达不成熟神经元标志物Tuj1,表达星形胶质细胞标志物GFAP,表达少突胶质细胞标志物galactocerebroside,以及谷氨酸能神经元标志物GluR-1,证明其具有多向分化潜能。结论本实验证明耳蜗前体细胞具有神经干细胞的多向分化潜能,为研究通过基因治疗的方法促进耳蜗神经细胞增殖提供了一种体外模型。

Objective To isolate, culture and identify the cochlear progenitor cells （CPCs）. Methods CPCs were isolated from P7 rat cochlea tissues and cultured. We demonstrated progenitor cells in postnatal rat cochleae and their potential to differentiate into multiple lineages using immunocytochemistry. Results After 1 day of culture, the acutely isolated cells formed floating otospheres. Most cultured cells expressed nestin, musashil and pax2 and incorporated BrdU after 7 days of culture, indicating that the cells in these spheres not only expressed the specific markers of stem cells but were also actively undergoing mitosis. The progenitor cell-derived differentiated cells expressed myosin VIIA, phalloidin, NeuN, Tuj1, GFAP, galactocerebroside and GIuR-1. The cells generated from the cochlea-derived spheres contained hair ceils, neurons, neuroglial cells, and glutamic neurons. Conclusion The progenitor ceils were present in the cochlea of newborn SD rats. They are capable of self-renewing and possess potential to differentiate into multiple lineages. This study provides a good in vitro model for studying the mechanisms controlling the dormant state of the CPCs in adults