鼻息肉成纤维细胞在常氧与低氧环境下的原代培养及鉴定

Primary culture and identification of fibroblasts cells from nasal polyps under normoxia and hypoxia

目的探讨鼻息肉成纤维细胞在常氧与低氧环境下的原代培养及鉴定，以备后续研究其生物学特性。方法取2012至2013年行鼻内镜下鼻息肉切除的息肉组织20例。标本放入4℃无菌的PBS溶液中，在超净台中反复冲洗、浸泡，剪成1-2mm大的组织块后，分成2份转入EP管中，分别用0．1％I型胶原酶和1mL的dispase Ⅱ酶消化过夜。次日取出，轻轻吹打，进行计数后，加入培养液（DMEM＋10％胎牛血清＋1×10^5U/L青霉素＋100mg/L链霉素），分别置于常氧、低氧条件下培养（常氧培养以空气作为混合气体，氧体积分数约为20％；低氧培养则以氮气作为混合气体，调节氧体积分数为5％。二者其他培养条件相同，均为37℃、含体积分数为5％的CO，和饱和湿度）。在倒置相差显微镜下观察细胞的形态、增殖及生长状况、有无污染。用CCK-8比色法检测细胞的增殖，描绘生长曲线。结果dispaseII酶消化组的细胞数目高于Ⅰ型胶原酶消化组，两组之间差异有统计学意义（P〈0．05）。鼻息肉成纤维细胞在常氧及低氧条件下培养的生长曲线无明显差异。结论dispaseⅡ酶消化的细胞数目较多，是一种较好的获得细胞的方法。在常氧及低氧条件下均可建立稳定、可靠的人鼻黏膜成纤维细胞原代培养模型，可为鼻息肉的相关研究提供良好的细胞系。

Objective To investigate the primary culture and identification of fibroblasts cells from nasal polyps under normoxia and hypoxia for further study on their biological features. Methods The specimens of nasal polyps （20 cases） endoscopicaIIy removed in our department from 2012 to 2013 were placed into 4 ℃ sterile phosphate-buffered sa line（PBS） solution supplemented with penicillin and streptomycin, repeatedly washed, immersed, cut into pieces, and tubed into two portions to EP, respectively with 0. l%collagenase Ⅰ and 1 mL of dispase Ⅱ overnight. The next day, the samples were removed, pipetted slightly to detach the epithelia, after to count, and added in broth （DMEM ＋ 10% fetal bovine serum ＋ 1 x 10^5 U/L penicillin ＋ 100 mg/L streptomycin）, respectively in normoxia and hypoxia conditions. Besides normoxia training with air as the mixed gas, oxygen volume fraction of about 20% ; hypoxia training with nitrogen as the mixed gas, regulating oxygen volume fraction of 5 %, the other culture conditions were the same with the volume fraction of 5% CO2 and saturated humidity at 37 ℃. Under an inverted phase microscope, the cells morphology, proliferation, growth and infection were observed. The proliferation of cells was tested by CCK-8. Results Overall improvement was observed in the number of cells in dispase II and was higher than that of type colla genase Ⅰ （P 〈 0.05 ）. Nasal polyp fibroblasts in normoxia and hypoxia condition training had no obvious difference. Conclusion Dispase Ⅱ can be used to obtain more cells. In normoxia and hypoxia condition, the above mentioned model is more suitable for primary culture due to its fast proliferation, stable and reliable cell supply for nasal polyps research.