大鼠耳蜗大上皮嵴细胞体外培养的实验观察  

In vitro culture of greater epithelial ridge cells from rat cochleae

作　　者：张媛[1] 孙建和[1] 胡吟燕[1] 郑贵亮[1] 翟所强[1] 

机构地区：[1]解放军总医院耳鼻咽喉科研究所

出　　处：《中华耳鼻咽喉头颈外科杂志》2007年第10期760-764,共5页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家自然科学基金(30440063);海外青年学者合作研究基金(30228029);北京市自然科学基金(7053076)

摘　　要：目的建立大鼠耳蜗大上皮嵴(greater epithelial ridge,GER)细胞体外培养体系,探讨其在离体培养条件下的生物学特性、生长模式及超微结构。方法取生后1d 大鼠耳蜗,利用机械分离和酶消化相结合的方法分离出纯 GER 细胞,分别于无血清和含10%胎牛血清的 DMEM 培基中进行培养,观察细胞生长特性,并对培养物进行溴脱氧尿苷(BrdU)、鬼笔环肽(phalloidin)、ZO1、钙视网膜蛋白(calretinin)及肌球蛋白 VIIa(myosin VIIa)免疫组化鉴定及扫描电镜观察。结果 GER 体外培养细胞呈现典型的上皮样多角形外观,细胞间连接紧密,具有明显的增殖能力,在含血清培养基中易形成贴壁细胞岛,而在无血清培养条件下则易形成悬浮细胞球。GER 细胞呈现 BrdU、phalloidin 以及ZO1染色的阳性反应,而 calretinin 及 myosinVIIa 则表现为阴性。扫描电镜显示 GER 细胞表面可见微绒毛和中心体,但未见毛细胞特异性结构纤毛束。结论 GER 体外培养细胞具有增殖能力,在不同培养条件下可分别呈现贴壁细胞岛及悬浮细胞球生长特性,GER 细胞表达上皮细胞来源标记物,但不表达毛细胞特异性标记物。Objective To establish in vitro culture systems of greater epithelial ridge （GER） cells from rat cochlear and to investigate the characterization, growth pattern and ultrastructure of GER cells. Methods Using a combinatorial approach of enzymatic digestion and mechanical separation to allow isolation and culture of GER cells from P1 rat cochleae. The dissociated GER cells were cultured in serumfree or 10% fetal bovine serum DMEM respectively. BrdU, phalloidin, ZO1, calretinin and myosinVIIa immunostaining and scanning electron microscope observation were performed in GER cell cultures. Results The dissociated GER cell cultures showed positive to ZO1 ,phalloidin and BrdU staining,but negative to myosin VIIa and calretinin. They assumed a polygonal morphology which was similar to epithelial cells and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serumfree medium. The GER cells presented significant ability to proliferate in both conditions. Scanning electron microscope showed that there was microvillus and centrebodies but not hair cell specific stereocilliary bundles on the surface of GER cultures. Conclusions The GER cell cultures showed significant ability to proliferate and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The dissociated GER cells expressed epithelial cell specific marker but not marker of hair cells.

关 键 词：耳蜗 细胞 培养的 细胞培养技术 

分 类 号：R764[医药卫生—耳鼻咽喉科]