胎鼠听囊细胞体外培养探究.docVIP

胎鼠听囊细胞体外培养探究 　　　作者：刘晖，程随涛，许珉，梁建民，马伟军 【关键词】 听囊细胞；干细胞；细胞增殖；细胞分化；胎鼠 【Abstract】 AIM: To find the origin of auditory cells in mammals and to study the proliferation and differentiation of auditory cell progenitors. METHODS: Otocyst epithelia surgically isolated from fetal rats were cultured and passaged. Proliferation and differentiation of cultured cells were observed using BrdU labeling to find the progenitors. Immunocytochemistry (Factin, cytokeratin and calretinin) was used to detect cell properties of cultured cells. RESULTS: Otocyst cells differentiated and proliferated and showed several morphotypes, mainly including epitheliallike cells, fibroblastlike and neuronlike cells. All of cells were labeled positive to Factin and cytokeratin. A subset of cells were labeled strong positive by calretinin in primary culture. But few passaged cells were positive to calretinin. BrdU labeling showed cultured cells could proliferate significantly. The morphological phenotype was retained throughout 8 passages during 4month culture. CONCLUSION: In natural culture condition in vitro, otocyst cells of fetal rats can differentiate or proliferate to immature hair cells. The study will provide some ideas and methods for further research about hair cell regeneration. 【Keywords】 otocyst cells; stem cells; cell proliferation; differentiation; fetal rats 【摘要】 目的： 了解哺乳动物听觉细胞的来源，探索听觉细胞前体细胞体外增殖和分化. 方法： 取孕鼠胚胎听囊细胞进行体外培养，用免疫细胞化学方法，分别对培养的细胞进行细胞角蛋白、F肌动蛋白和calretinin染色观察细胞特征；BrdU染色观察细胞增殖. 结果：培养的细胞在体外进行分化和增殖，细胞形态主要有上皮细胞形态、成纤维细胞形态和神经细胞形态3种. 对细胞角蛋白和F肌动蛋白这两种上皮细胞的标志染色呈阳性. 在原代培养就出现calretinin染色明显阳性的细胞. 传代后结果仍为阳性着染，但强度减弱. BrdU染色显示培养细胞具有明显的增殖活性. 本研究共传8代，历时4 mo，细胞形态维持稳定. 结论： 胎鼠听囊细胞在体外自然培养条件下可以分化或者增殖为未成熟毛细胞. 本研究为探索刺激听觉毛细胞再生的治疗方法提供研究思路和方法. 【关键词】 听囊细胞；干细胞；细胞增殖；细胞分化；胎鼠 由噪声、耳毒性药物等引起的耳蜗毛细胞损害而致的感音神经性耳聋是耳鼻咽喉科难治之症. 如果能够建立一种细胞系，而这种细胞系又具有分化为听觉毛细胞的潜能，不但有利于听力学研究，也有利于进行药物的发展及毛细胞生物学方面的研究. 我们对胎鼠听囊细胞用自然培养方法进行体外培养，并应用免疫细胞化学方法，对培养细胞进行了染色标记和观察. 1材料和方法 1.1材料选用怀孕14 d成年健康小鼠1只，灰色，体质量0.15 kg. 耳廓反射正常，鼓膜正常. 细胞培养基DMEM（Gibco公司）；