2种气管黏膜上皮细胞体外培养技术的研究

Research on two culture methods for bronchia epithelia

目的为以猪气管黏膜上皮为细胞模型的研究奠定物质基础,进一步探讨猪气管黏膜上皮细胞的体外传统培养和气液界面培养技术,从而使2种培养技术优势互补。方法对猪气管上皮分离、纯化、培养和传代,并探索上皮细胞最佳冻存复苏条件;复苏后的气管上皮细胞进行气液界面培养,绘制细胞生长曲线和观察细胞纤毛生发情况。利用免疫组化法鉴定上皮细胞。结果4步纯化法可以得到高纯度的气管上皮细胞。使用胎牛血清、DMEM/F12培养液和DMSO的体积分数为50%、40%和10%的冻存体系保存的气管上皮,复苏后细胞存活率平均可达89%。优化后的传统方式培养的上皮细胞可连续传代到第8代,但从第2代开始便观察不到纤毛,转换成气液界面连续培养2代后重新生发纤毛,细胞存活期延长。免疫组化结果显示分离培养细胞为上皮细胞。结论成功建立了2种猪气管上皮细胞培养技术,并找到适宜的气管上皮细胞冻存条件,节省了不断原代取材的成本和时间,并成功实现传统培养细胞冻存复苏后很快适应气液界面的培养并恢复细胞的天然结构,为猪气管黏膜上皮相关研究提供丰富的细胞来源。

Objective To explore two technologies of separation and culture for pig trachea-bronchia epithelia： traditional culture and air-liquid interface culture,look for appropriate freezing and anabiosis conditions of the epithelia and combine advantages of the two culture technologies.Method Pig trachea-bronchia epithelia was separated,purified and kept passage.The livability of descended epithelia was calculated by the assay of freezing and anabiosis to look for best freezing and anabiosis conditions.After anabiosis the epithelia were cultured by air-liquid method and its growth characteristics and the appearance of the cells′ cilia were observed.The growth curve was also made.Finally the test of keratins of pig trachea-bronchia epithelia was done to identify the epithelia.Result Through four-step purification highly pure epithelia were gotten.The average epithelia livability was 89% after anabiosis,which was freezed with a system including 50% of Fetal Serum,40% of DMEM/F12 and 10% of DMSO.The epithelia passage number was eight with the traditional culture and after the second generation the epithelia cilia became invisible.The cilia of some epithelia cultured by air-liquid method came into being after continuously descended twice and the epithelia＇s suvival time was also prolonged.The result of the test of keratins indicated the cultured cells were epithelia.Conclusion We have successfully established two culture technologies and found the appropriate freezing and anabiosis technology for pig trachea-bronchia epithelia.The epithelia by traditional culture,after freezing and anabiosis,can successfully adapt to air-liquid culture and recover their natural morphological characteristics.The advantages of the two culture technologies have been successfully combined,which provide a rich cell source for researches of pig trachea-bronchia epithelia.