应用嗜热菌蛋白酶快速分离活性纯椭圆囊感觉上皮细胞及其意义

Isolation of Pure Living Utricular Sensory Epithelial Cell by Use of Thermolysin

目的建立纯椭圆囊感觉上皮细胞(pure utricular sensory epithelial cell,PUSEC)的快速分离方法,为内耳细胞培养及毛细胞再生等机制研究提供大量的活性细胞。方法通过对出生5天大鼠PUSEC的形态、生长特征的观察,采用透射电镜,紧密联接蛋白(ZO-1)、细胞角蛋白、波形蛋白免疫荧光细胞化学、RT-PCR检测毛细胞的特征性标志物Calretinin、Brn3.a mRNA的表达等方法鉴定PUSEC来源。结果PUSEC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。可见由成百个PUSEC包绕液体而成的dome。PUSEC表达ZO-1和细胞角蛋白,不表达波形蛋白,表达毛细胞的特征性标志物Calretinin、Brn3.a的mRNA。结论PUSEC来源于椭圆囊感觉上皮;纯椭圆囊感觉上皮细胞成功分离为内耳细胞培养及毛细胞再生机制的研究提供大量的活性细胞;成功分离的关键成份是嗜热菌蛋白酶。

Objective To study rapidly collected high - purity samples of living hair cells and supporting cells from inner ear and to investigate the mechanisms of hair cell generation or regeneration. Methods Utrieular sensory epithelium of postnatal day 5 （P5） rats was isolated by mechanical dissociation. The explants were digested by thermolysin, then transferred to an aliquot containing trypsin and eollagenase for incubation to harvest the pure utrieular sensory epithelial cell （PUSEC）. PUSEC was cultured in Dulbeceo modified Eagle medium（DMEM）, and observed daily under inverted microscope.The uhrastrctural examination with transmission electron microscope, immunofluorescenee method with ZO- 1, eytokeratin , vimentin; reverse transcription PCR with mRNA of Calretinin, Brn3. a were used to identify the origin and characterization of PUSEC. Results PUSEC showed a large, flat, polygonal epithelial morphotype with big, round neuclei. PUSEC grew into monolayer, ＂cobblestone- like＂ apearance. Some cells showed ＂dome＂ formation,probably due to fluid collection underneath the cell monolayer.The PUSEC expressed ZO-1 and cytokeratin 18, without vimentin expression, and had rich microvilli and complex tight junction, which indicated the epithelium origination of PUSEC. Coexpressed the mRNA of Calretinin, Brn3. a of hair cell characteristic markers identified PUSEC were derived from utricular sensory epithelium. Conclusion Isolation method of pure living utricular sensory epithelial cell of the rats is successfully established. It is suggested that the key ingredient is thermolysin.