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目的研究新生大鼠耳蜗内是否存在增殖细胞,以及该增殖细胞能分化为何种细胞。方法分离新生 SD 大鼠耳蜗细胞并进行培养,加入5-溴-2-脱氧尿嘧啶(5-bromo-2-demoxyuridine,BrdU)检测细胞的增殖状态,通过免疫荧光鉴定细胞球及分化细胞的性质。分离48个耳蜗 Corti 器,采用数字表格法随机分成4组进行细胞培养:对照组、表皮生长因子(epidermal growth factor,EGF)组、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)组和 EGF+bFGF 组,每组12个,定量计数单个耳蜗培养细胞球的数量,结果进行方差分析。采用透射和扫描电镜观察细胞球的超微结构。结果耳蜗 Corti 器细胞培养可见细胞球形成,90.1%球内细胞 BrdU 表达阳性,且大部分细胞巢蛋白表达阳性。分化细胞表达毛细胞标志物肌球蛋白7A、espin 及鬼笔环肽阳性,神经元标志物神经丝蛋白(neurofilament M,NF-M)表达阳性,并且可见肌球蛋白7A 和 BrdU,espin 和 BrdU,以及 NF-M和 BedU 双标阳性的分化细胞。对照绀平均(±s,以下同)每个耳蜗 Corti 器的增殖细胞数量为(45.3±23.0)个,EGF 组(86.2±34.1)个,bFGF 组(96.5±33.6)个,EGF+bFGF 组(131.2±47.0)个。除EGF 组和 bFGF 组之间 P>0.05外,其余各组之间 P 值均<0.05,差异具有统计学意义。扫描和透射电镜下细胞球内细胞呈网球形,大小均匀一致,表面具有众多微绒毛。胞质内有丰富的内质网、微管、微丝和线粒体等细胞骨架结构和细胞器,相邻细胞之间可见紧密连接、桥粒与缝隙连接。结论大鼠耳蜗内存在增殖细胞,可分化为具有纤毛样结构的毛细胞及神经元。EGF 和 bFGF 均可诱导增殖细胞分裂增殖。该增殖细胞的超微结构显示其具有早期幼稚细胞发育的特征。
Objective To investigate the existence of proliferating cells in the cochlea of newborn rats and the differentiation of proliferating cells into those cells. Methods The cochlear cells of neonatal SD rats were isolated and cultured, and the proliferation status of the cells was detected by using 5-bromo-2-deoxyuridine (BrdU). The characteristics of the spheres and differentiated cells were identified by immunofluorescence . Forty-eight cochlear Corti devices were isolated and divided into four groups according to the digital table method: control group, epidermal growth factor (EGF) group, basic fibroblast growth factor (bFGF) group And EGF + bFGF group, 12 in each group. The number of single cultured cochlear cells was counted and the results were analyzed by ANOVA. Transmission electron microscopy and scanning electron microscopy of the ultrastructure of the cell. Results Cochlear cells were cultured in Corti cells. Spherical cells formed in 90.1% of the cells, and BrdU in most of the cells showed positive expression of Nestin. Differentiated cells expressed the hair cell markers myosin 7A, espin and phalloidin, positive neuronal markers neurofilament M (NF-M), and showed that myosin 7A and BrdU, espin and BrdU , And differentiated cells with double positive for NF-M and BedU. (45.3 ± 23.0), EGF (86.2 ± 34.1), bFGF group (96.5 ± 33.6), EGF + bFGF group ( 131.2 ± 47.0). Except P> 0.05 between EGF group and bFGF group, P values of other groups were all <0.05, the difference was statistically significant. Scanning and transmission electron microscopy within the cytoplasmic cells were spherical, uniform size, the surface has a large number of microvilli. The cytoplasm is rich in endoplasmic reticulum, microtubules, microfilaments and mitochondria and other cytoskeleton structures and organelles, adjacent cells can be seen in close connection, desmosomes and gap connection. Conclusion There are proliferating cells in the rat cochlea, which can differentiate into ciliated hair cells and neurons. Both EGF and bFGF can induce proliferation and proliferation of proliferating cells. The ultrastructure of this proliferating cell shows that it has the characteristics of early naive cell development.