大鼠椭圆囊再生毛细胞前体细胞来源的初步研究  

The Origination of Progenitor Cells of Utricular Regenerative Hair Cells in Rat

作　　者：刘俊[1] 孔维佳[2] 赵荣祥[1] 

机构地区：[1]浙江省中医院耳鼻咽喉科,杭州310006 [2]华中科技大学同济医学院附属协和医院耳鼻咽喉科

出　　处：《听力学及言语疾病杂志》2009年第5期465-469,共5页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金(39925035);国家杰出青年基金(39925035);卫生部临床学科重点项目(2001321)基金资助

摘　　要：目的探讨哺乳动物椭圆囊再生毛细胞前体细胞的可能来源。方法取出生1天的大鼠的椭圆囊,经嗜热菌蛋白酶(thermolysin)处理,消化法进行原代培养。在倒置显微镜下观察椭圆囊感觉上皮细胞(utricular sensory epichelial cell,USEC)的形态、生长特征;透射电镜观察、免疫细胞化学法检测上皮细胞角蛋白18、波形蛋白等鉴定USEC上皮细胞来源。免疫细胞化学法、RT-PCR技术检测支持细胞标记物p27kiplmRNA及毛细胞的特征性标记物Brn3a、Calretinin及AchRa9、MyosinⅦa mRNA的表达。结果原代培养的USEC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。可见由数百个USEC包绕液体而成的dome(穹窿样)结构,表达细胞角蛋白,不表达波形蛋白,微绒毛丰富,细胞间连接紧密,提示其上皮来源。原代培养的USEC表达支持细胞标记物p27kiplmRNA及毛细胞的特征性标志物Brn3a、Calretinin及AchRa9、MyosinⅦa mRNA,表明培养的USEC可能来源于支持细胞并具有毛细胞的特性。结论原代培养的USEC能产生毛细胞样细胞且表达支持细胞标记物,表明其来源为支持细胞。椭圆囊感觉上皮的支持细胞可能为毛细胞再生的前体细胞之一。Objective To investigate the cell origination of progenitor cells of the mammalian regenerative hair cell. Methods Utricular sensory epithelial of postnatal day 1 wister rats was isolated by mechanical dissociation. The explants were digested by thermolysin,then were transfrred to an aliquot containing 0. 125% trypsin and 0. 125% collagenase for incubation to harvest the pure utricular sensory epithelial cell（USEC）. USEC were cultured in Dulbecco Modified Eagle Medium（DMEM）, and observed daily by inverted microscope. Immunocytochemical staining with cytokeratin 18 , vimentin, Brn3a and Calretinin, ultrastrctural examination with transmission electron microscope, and reverse transcription PCR with mRNA of p27^kipl , AchRa9 and Myosin Ⅶ a. The markers of hair cells and supporting cells were used to identify the origination and characteriztion of USEC. Results USEC showed a large, flat,polygonal epithelial morphotype with big,round neuclei. USEC monolayer showed ＂cobblestone- like＂ appearance. Some cells showed ＂Dome＂ formation, probably due to fluid collection underneath the cell monolayer. USEC expressed cytokeratin 18 but did not express vimentin,and had rich microvilli and complex tight junction, which indicated the epithelial origination of USEC. USEC expressed Brn 3a,calnevimin and mRNA of p27^kipl,AchRa9 ,myosin Ⅶ, Conclusion USEC coexpressed the characteristic markers of the hair cell and supporting cells. This indicates that the culture cells may represent progenitor cell of hair cell and the progenitor cell may come from supporting cells.

关 键 词：椭圆囊 感觉上皮 再生 毛细胞 

分 类 号：R339.16[医药卫生—人体生理学]