大鼠血管纹边缘细胞永生细胞系的建立

Establishment of immortal marginal cell line of the rat stria vascularis

目的建立大鼠血管纹边缘细胞(Marginal Cells,MC)的永生细胞系,为进一步研究MC的功能打下基础。方法取3～5日龄大鼠的耳蜗侧壁组织进行MC的原代培养,经选择性消化、差异性贴壁等手段纯化MC后,将MC和SV40在无血清DMEM条件下共培养三天,经2%新生牛血清选择培养三天,通过形态学的变化及增殖能力的变化粗筛出MC系;通过观察MC系的形态、生长特征,细胞角蛋白18、波形蛋白的免疫细胞化学,RT-PCR检测SV40T抗原、IsK,细胞记数法及流式细胞术分析其生长情况及染色体倍型,软琼脂培养、裸鼠致瘤实验等鉴定MC-C3系。结果MC-C3系基本保持了原代MC的表型特征:多角形外观、细胞间连接紧密,单层时呈“铺路石样”外观,dome形成能力等形态、生长特征;细胞角蛋白18、波形蛋白表达双阳性;不同世代的MC-C3均有SV40T抗原、IsK的表达。MC-C3为二倍体细胞。软琼脂培养和裸鼠致瘤实验阴性。该系目前已培养5月,超过20代,传代时间为4～5天,传代比例为1:2～3。液氮中保存有不同世代的细胞。结论本实验成功的建立了大鼠边缘细胞的永生细胞系,初步鉴定表明MC-C3基本保留了原代边缘细胞的表型特征,又不具有瘤细胞的特征。为边缘细胞功能的研究,特别是电生理、分子生物学方面的研究打下了良好的基础。

Objective To establish marginal cell （MC） immortal line originated from rats. Methods The lateral wall tissues of rat pups＇ cochleae Were excised as explants for the primary culture of marginal cells. The purified MC was co-cultured with SV40 in serum-free DMEM for three days, then was chosen by 2% new born calf serum （NBCS） in DMEM for another three days. One population of MC was selected as the permanent MC line many times later. Immunocytochemical staining for cytokeratin 18 and vimentin, uhrastructural examination, reverse transcription PCR amplifying eDNA of MC T-antigen gene, the marker of immortalization, and IsK-protein gene, the unique marker of MC, flow cytometric analysis of MC＇s DNA content, injection of the MC to nude mice and sofi-agar-medium culture were used to identify the origin and characterization of MCs. Results MC-C3 may grow into monolayer with ＂cobblestone-like＂ appearance and dome formation ability. MC-C3 co-expressed cytokeratin 18 and vimentin, showing its epithelial origin. mRNAs of SV40 large T antigen and IsK protein existed in the MC-C3. Flow cytometric analysis of this cell line＇s DNA content was diploid. The test for neoplasminducing ability of MC-C3 was negative. It has been passaged 21 time. The passage interval was 4-5 days and the passage ratio was 1：2-3. Different passages of MC-C3 were kept at -196℃. Conclusion The MC line （MC-C3） has been successfully established. It kept the phenotype characterization of the primary MC, and did not contain the neoplasm behavior. So it is suitable for study of the MC function, especially in electrophysiological and biomolecular aspects.