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目的:建立大鼠耳蜗感觉上皮细胞(CSEC)的原代培养体系,为研究毛细胞再生的分子机制提供大量来源一致的细胞。方法:取出生1d(P1)的大鼠耳蜗感觉上皮(Corti器)作组织块培养,选择性消化抑制成纤维样细胞(FLC)生长;应用有限稀释法纯化CSEC,倒置显微镜下观察CSEC的形态、生长特征;应用细胞角蛋白18、波形蛋白、Brn3.a和Calretinin的免疫细胞化学及透射电镜等进行鉴定。BrdU标记CSEC核DNA观察CSEC有丝分裂活动。结果:培养第2天CSEC从组织块迁移出来,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,形成单层时呈“铺路石样”外观;FLC呈梭形“鱼群样”包绕CSEC生长,增殖速度快于CSEC。由纯化的CSEC构成的上皮单层可见由成百个CSEC包绕液体形成的dome。CSEC表达毛细胞的特征性标志物,证实培养的CSEC具有毛细胞前体细胞的特征;并通过有丝分裂活动产生新的细胞即毛细胞样细胞。结论:体外培养的CSEC表达毛细胞特征性标志物,具有毛细胞前体细胞的特征。CSEC原代培养体系的建立,为进一步探讨毛细胞再生的分子机制提供了实验条件。
OBJECTIVE: To establish a primary culture system of rat cochlear sensory epithelial cells (CSEC) and provide a large number of cells with consistent sources for studying the molecular mechanism of hair cell regeneration. Methods: Rat cortical sensory epithelium (Corti) of primary 1d (P1) was taken as a tissue culture block and selectively digested to inhibit the growth of fibroblastoid cells (FLC). CSECs were purified by limiting dilution method and the morphology of CSEC was observed under inverted microscope , And their growth characteristics. Immunocytochemistry and transmission electron microscopy were used to identify cytokeratin 18, vimentin, Brn3.a and Calretinin. BrdU labeling CSEC nuclear DNA to observe mitotic activity of CSEC. Results: On the second day of culture, CSEC migrated out of the tissue mass with a flat, polygonal, large nucleus and round epithelial morphology. The cells were tightly connected to form a “paving stone-like” appearance when the monolayer formed. “Fish-like” around CSEC growth, proliferation faster than CSEC. The epithelial monolayer made of purified CSEC shows a dome formed of hundreds of CSEC-enveloped liquids. CSECs express characteristic markers of hair cells, demonstrating that cultured CSECs possess the characteristics of hair cell precursors and produce new cells, ie, hair cell-like cells, through mitotic activity. CONCLUSION: CSEC cultured in vitro expresses the characteristic markers of hair cells and possess the characteristics of hair cell precursors. The establishment of the primary culture system of CSEC provided the experimental conditions for further exploring the molecular mechanism of hair cell regeneration.