大鼠椭圆囊感觉上皮细胞的原代培养及其意义  

Establishment and characterization of primary culture of rat utricular sensory epithelial cells

作　　者：刘俊[1] 孔维佳[1] 张丹[1] 张亚民[1] 

机构地区：[1]武汉华中科技大学同济医学院附属协和医院耳鼻喉科,武汉430022

出　　处：《中华耳科学杂志》2006年第2期123-128,共6页Chinese Journal of Otology

基　　金：国家自然科学基金(No:39925035);教育部高等学校骨干教师计划基金(No:200065);教育部高等学校博士学科点专项基金(No:2002173);国家十五科技攻关项目(我国耳神经疾病早期干预措施及规范化治疗方案研究;2004BA720A18-02)等基金项目。

摘　　要：目的建立大鼠椭圆囊感觉上皮细胞(utricularsensoryepithelialcell,USEC)的原代培养体系,为研究毛细胞再生机制提供大量来源一致的细胞。方法取出生1天(postnatalday1,P1)大鼠的椭圆囊感觉上皮,嗜热菌蛋白酶(thermolysin)消化处理,获得纯椭圆囊感觉上皮,再采用消化法进行原代培养,培养基为DMEM。从第2天开始每日用倒置显微镜观察、照相,对USEC的形态、生长特征进行观察;透射电镜观察;应用细胞角蛋白18及波形蛋白免疫细胞化学方法鉴定USEC来源;应用免疫细胞化学及RT-PCR分别检测毛细胞的特征性标志物calretinin、Brn3.a和AChR!9、myosinⅦamRNA的表达。结果原代培养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,可见由成百个USEC包绕液体而成的dome。原代USEC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,提示其上皮起源;表达毛细胞的特征性标志物Brn3.a、calretinin及AChR!9、myosinⅦamRNA,表明培养USEC具有毛细胞前体细胞的特征。结论原代培养的USEC表达毛细胞的特征性标志物,具有毛细胞前体细胞的特征。USEC原代培养体系的建立,为进一步探讨毛细胞再生的机制提供充足的细胞来源。Objective To investigate the mechanisms of hair cell generation and regeneration, a primary culture system of utricular sensory epithelial cells of the rat was established. Methods Utricular sensory epithelia of postnatal day 1 rats were isolated and digested by thermolysin, then were transferred to a solution containing 0.125% trypsin and 0.125% collagenase for 8 min at 37℃ to harvest the pure utricular sensory epithelial cells（ USECs ）. USECs were cultured in Dulbecco＇s modified Eagle＇s medium （DMEM）, and observed daily by inverted microscope. The culture medium was changed twice a week. USECs were identified by immunocytochemical staining for cytokeratin 18, vimentin, calretinin and Brn3a, and by reverse transcription PCR for AChR α9 and myosin Ⅶa mRNA expressions. USECs were also assessed by transmission electron microscopy. Results The USECs showed a large flat, polygonal shape with big round nuclei. USECs formed monolayer and exhibited a ＂cobblestone-like＂apearance. Some cells showed ＂dome＂ formation, probably due to fluid collection underneath the cell monolayer. The cultured USECs expressed cytokeratin 18, but not vimentin, and had abundant microvilli and complex tight junctions, indicating epithelial origin. Coexpression of calretinin, Brn3a and mRNAs of AChR α and myosin Ⅶa, four characteristic hair cell markers, supported the view that the cultured cells may represent rat progenitor hair cells. Conclusion The primary culture system of utricular sensory epithelial cells of the rat was established. The USECs coexpressed the characteristic hair cell markers, suggesting that the cultured cells may represent progenitor cells for the rat hair cell. It should be a suitable model for in-depth investigations of the mechanisms of hair cell generation and regeneration.

关 键 词：椭圆囊 感觉上皮 原代培养 再生 毛细胞 

分 类 号：Q344[生物学—遗传学] Q437