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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 an in vitro culture system of large epithelial ridge (GER) cells in rat cochlea and investigate its biological characteristics, growth pattern and ultrastructure under in vitro culture conditions. Methods The cochleas of rats were sacrificed on day 1 of postnatal day. Pure GER cells were isolated by mechanical and enzymatic digestion. The cells were cultured in serum-free and DMEM medium containing 10% fetal bovine serum to observe the growth characteristics of cells The culture was identified by BrdU, phalloidin, ZO1, calretinin and myosin VIIa immunohistochemistry and scanning electron microscopy. Results GER cells cultured in vitro showed typical epithelial-like polygonal appearance with tight intercellular junctions and obvious proliferative ability. Adherent cells were easily formed in serum-containing medium, while suspension cells were easily formed in serum-free medium ball. GER cells showed the positive reaction of BrdU, phalloidin and ZO1 staining, while calretinin and myosinVIIa showed negative. Scanning electron microscopy showed microvilli and centrosomes on the surface of GER cells, but no hair cell-specific structural cilia bundles were observed. Conclusions GER cells have proliferative ability in vitro. Under different culture conditions, GER cells express epithelial cell-derived markers but do not express hair-cell-specific markers.