哺乳动物耳蜗感觉上皮细胞长期培养体系的建立及临床意义

Establishment of long-term culture system of mammalian cochlear sensory epithelial cells

目的建立哺乳动物耳蜗感觉上皮细胞（CSEC）长期培养体系，为内耳毛细胞再生、分子及基因研究提供细胞来源。方法取1d龄wistar大鼠耳蜗感觉上皮作组织块培养，有限稀释法纯化并得到CSEC单克隆细胞系。胰蛋白酶＋胶原酶消化培养传代，传25代。取第25代CSEC，在倒置显微镜、透射电镜下对CSEC的生长特征、形态结构进行观察；免疫细胞化学检测细胞角蛋白18、波形蛋白，鉴定CSEC细胞来源。免疫荧光细胞化学、RT—PCR检测毛细胞标志物Brn．3a、Calretinin及AchRa9、MyosinVllamRNA的表达，鉴定CSEC特征。结果第25代CSEC均呈扁平、多角形、核大而圆的上皮细胞形态，细胞之间连接紧密，单层时呈“铺路石样”外观，可见“dome”结构；其可表达细胞角蛋白和不表达波形蛋白，微绒毛丰富，细胞间连接紧密，提示其为上皮细胞来源：并可表达毛细胞特征性标志物Brn3．a、Calretinin及AchRa9、Myosin Vlla mRNA。结论本实验成功建立了哺乳动物CSEC长期培养体系。长期培养的CSEC性状未发生明显改变，具有毛细胞前体细胞的特征。这为毛细胞再生的机制及内耳分子、基因研究提供了稳定的细胞来源。

Objective To establish a long-term culture system of mammalian cochlear sensory epithelial cells. Methods Cochlear sensory epithelial cells （CSECs） were isolated by mechanical dissociation from rats aged ld, and cultured in Dulbecco modified Eagle medium （DMEM）.Cell clones of CSEC were obtained by pre-diluted seeding and the pure cochlear sensory epithelial cells were harvested. Pure CSEC were cultured for 25 passages. CSECs of the 25 passages were observed by inverted microscope and transmission electron microscope. The cytokeratin 18 and vimentin were detected by immunocytochemical method; Bm.3a and Calretinin were examined by immunofluorescence method; the mRNAs of AchRa9 and Myosin VU a were measured by reverse transcription PCR. The markers of supporting cells and markers of hair cells were used to identify the characteristics of CSECs. Results CSECs were cultured for 25 passages in 6 months. CSECs of the 25 passages showed a large, flat, polygonal epithelial appearance with big, round neuclei. CSECs of the 25 passages grew into monolayer with cobblestone-like appearance; while some cells showed ＂Dome＂ formation. The CSECs of the 25 passages expressed cytokeratin 18, but no vimentin. The cells had rich microvilli and complex tight junction, which indicated the epithelial origin of CSECs. The cells were co-expressed with Brn3.a,Calretinin and mRNA of AchR a9, Myosin Vlla, which represented rat hair cell progenitor. Conclusion The long-term culture system of rat cochlear sensory epithelial cells was successfully established, which provides the cell sources for study on generation or regeneration of hair cells in inner ear.