摘要
目的通过体外定向诱导耳蜗前体细胞分化,明确耳蜗前体细胞的位置取材,来源及增殖分化特性,建立完善的耳蜗前体细胞的培养体系。方法取耳蜗的Corti器位置,利用无血清培养和单细胞克隆技术,进行细胞培养,在相差显微镜下观察细胞形态及生长状况,并用神经巢蛋白(nestin)、溴脱氧尿嘧啶(Brdu)、MyosinⅦA、P27KIP1等免疫荧光方法,鉴定耳蜗前体细胞和由耳蜗前体细胞分化而来的内耳毛细胞和内耳支持细胞。同时进行细胞计数。结果从新生大鼠耳蜗分离的组织,经原代和传代培养后,可获得大量未分化,悬浮生长的Nestin阳性细胞团,并具有增殖的能力,诱导分化后的细胞经免疫荧光双标实验可表达Brdu、MyosinⅦA和P27KIP1阳性。流式细胞仪细胞计数后,发现前体细胞向内耳毛细胞诱导分化的数量较少,向支持细胞分化的较多。结论从新生大鼠耳蜗分离的细胞是具有自我更新和分化潜能的前体细胞,可诱导分化为内耳毛细胞和内耳支持细胞。
Purpose The cochlea tissues of neonatal rat were dissociated mechanically and enzymatically to observe the characteristics of proliferation and induced differentiation of the organ of corti tissues progenitor cells. Methods Methods of serum-free incubation and single-cell cloning technique were applied to culture the cochlea progenitor cells and observe the growth characteristic of the progenitor cells. Immunofluorescence and flow cytometry were used to detect the results of progenitor cell characters. The expression of nestin, Brdu, MyosinⅦ A and P27^KIP1 were examined in culture. Results The dissociated cells from the newborn rat organ of corti developed into much of indiffrentiated otopheres of nestin ( + ) cells when plated on a non-adherent substratum. Within cultured otospheres, brdu( + ) cells were rapidly give rise to newly formed Myosin Ⅶ A( + ) hair cells, Myosin Ⅶ A( + ) hair cells had incorporated Brdu and P27^KIP1 demonstrating that the cochlea progenitor cell could differentiate into hair cells and supporting cells. The nrmber of suppoting cells is much more than hair cells afer indudced differentiation by flow cytometry. Conclusions The dissociated cells from the newborn rat organ of corti were progenitor cells and have the capacity for self-renewed, and form spheres that express nestin ( + ). Brdu( + ) makers, in culture could give rise to newly formed Myosin Ⅶ A( + ) hair cells and supporting cells.
出处
《复旦学报(医学版)》
CAS
CSCD
北大核心
2006年第5期601-605,共5页
Fudan University Journal of Medical Sciences
基金
江苏省教育厅项目
江苏省高校自然科学基金项目(05KJD310156)
南京中医药大学青年科技创新基金项目(05XZR02)资助
关键词
耳蜗前体细胞
内耳毛细胞
内耳支持细胞
诱导分化
cochlea progenitor cells
hair cells
supporting cells
induced differentiation