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目的:建立椭圆囊感觉上皮细胞(USEC)长期培养体系,为內耳毛细胞再生研究提供稳定的活性细胞。方法:取1d龄wistar大鼠,在显微镜下取出椭圆囊上皮,嗜热菌蛋白酶处理,获得具活性的纯椭圆囊感觉上皮,胰蛋白酶+胶原酶消化,培养传代,传25代。取第25代USEC在倒置显微镜、透射电镜下对USEC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、Brn3.a及Calretinin的表达。RT-PCR检测毛细胞的特征性标记物AchRa9、MyosinⅦamRNA的表达。结果:USEC细胞培养达6个月传25代,第25代USEC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,形成单层时均呈“铺路石样”外观,可见dome结构。该细胞表达角蛋白18和不表达波形蛋白,微绒毛丰富,细胞间连接紧密,提示其上皮起源。第25代USEC表达毛细胞的特征性标志物Brn3.a、Calretinin及AchRa9、MyosinⅦamRNA,表明长期培养USEC仍具有毛细胞的前体细胞的特性。结论:本实验成功建立了USEC长期培养体系。长期培养的USEC性状未发生明显改变,表达上皮细胞及毛细胞的特征性标志物,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 the oval sac sensory epithelial cells (USEC) to provide stable active cells for hair cell regeneration in the inner ear. Methods: Wistar rats (1-day-old) were taken out, and the oval capsule epithelium and thermolysin were removed under the microscope to obtain the active pure oval sac sensory epithelium. Trypsin + collagenase was digested and passaged for 25 passages. The 25th generation of USEC was used to observe the growth characteristics and morphology of USEC under inverted microscope and transmission electron microscope. The expressions of cytokeratin 18, vimentin, Brn3.a and Calretinin were detected by immunocytochemistry. RT-PCR detection of hair cells characteristic markers AchRa9, Myosin VIIamRNA expression. Results: USEC cells were cultured for 6 months and reached the 25th generation. The 25th generation of USEC showed a flat, polygonal, large nucleus and round epithelial cell morphology with close connection between cells. "Appearance, showing the dome structure. The cells expressed keratin 18 and did not express vimentin, rich in microvilli, the cell junction closely, suggesting that the epithelial origin. The 25th generation of USECs expressed Brn3.a, Calretinin and AchRa9, MyosinⅦamRNA, which are characteristic markers of hair cells, indicating that long-term culture of USEC still possesses the characteristics of hair cell precursor cells. Conclusion: This experiment successfully established the USEC long-term culture system. Long-term cultured USEC traits did not change significantly, the expression of epithelial cells and hair cells characteristic markers, with hair cell precursor cells. This provides a stable source of cells for the mechanism of hair cell regeneration and the study of inner ear molecules and genes.