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目的:建立成年大鼠耳蜗血管纹缘细胞(MC)的体外培养体系。方法:采用活体组织分离及培养技术,获取培养的大鼠耳蜗MC,透射电镜观察细胞超微结构,免疫组织化学法检测上皮细胞标志性中间丝角蛋白的表达及纯度,RT-PCR检测MC特征性的中间丝角蛋白mRNA的表达。结果:体外培养的MC呈不规则多角形,细胞增殖相互连接,紧密排列呈现培养上皮细胞典型的“铺路石”样外观,并可见由数百个MC密集生长组成的折光性强的球形结构(dome);透射电镜观察可见细胞周围有微绒毛样结构;免疫细胞化学检查,纯化后95%以上的MC细胞质呈桔黄色,而对照组无此着色。RT-PCR扩增出标志中间丝角蛋白基因表达的PCR产物,条带位于300~400 bp,与目的条带(368 bp)相符。结论:采用组织块培养技术成功建立成年大鼠耳蜗血管纹MC的体外培养体系,为进一步研究成熟期MC的功能提供合适的细胞模型。
Objective: To establish an in vitro culture system of adult rat cochlea vascular borderline cells (MC). Methods: The cultured rat cochlea MCs were obtained by living tissue isolation and culture techniques. The ultrastructure of the cells was observed by transmission electron microscopy. The expression and purity of landmark intermediate filament keratin in epithelial cells were detected by immunohistochemistry. The MC characteristics were detected by RT-PCR Sex intermediate silk fibroin mRNA expression. RESULTS: The MCs cultured in vitro showed irregular polygons, and the cell proliferation was connected with each other. The close-packed arrangement showed the typical “paving stone” appearance of cultured epithelial cells. (Dome); microvilli-like structures around the cells were observed by transmission electron microscopy; more than 95% of the cytoplasm of MC was orange after immunocytochemistry, but not in the control group. RT-PCR amplified the PCR product which indicated the expression of intermediate silk keratin gene. The band ranged from 300 bp to 400 bp, which was consistent with the target band (368 bp). CONCLUSION: The in vitro culture system of adult rat cochlea stria vascularis MC was successfully established by tissue culture technique, which provided a suitable cell model for further study on the function of MC in mature stage.