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目的:探讨甲床上皮细胞体外培养及传代的方法。方法:实验于2004-10/2005-04在吉林大学第一医院中心实验室完成。①标本消毒后,切取甲床并剪碎。②2.5g/L胰蛋白酶+3g/L乙二胺四乙酸1∶1混合液,37℃下消化重复3次,获得细胞悬液。③离心两次,清洗细胞,加入无血清角化细胞培养液制成细胞悬液,计数后接种于25mm培养瓶中。④37℃,体积分数为0.05的CO2、饱和湿度的培养箱内培养,每2天换液1次。⑤至单层细胞铺满近培养瓶底的80%以上时进行传代。⑥第3代细胞检测项目:倒置显微镜下观察细胞生长情况并摄片;细胞计数,绘制生长曲线;鼠抗人角蛋白17抗体做免疫细胞化学。结果:①细胞生长情况:倒置显微镜观察接种后的细胞12h后,大部分贴壁,11d左右时细胞生长密度可达瓶底的70%～80%,呈铺路石样排列。②免疫细胞化学鉴定结果:培养的细胞70%以上特异性角蛋白17抗体染色阳性。③生长曲线和群体倍增时间:接种后一两天细胞数减少,自第3天起细胞数迅速增多,至9～12d达高峰。对数期细胞群体倍增时间为4.46d。结论:利用酶消化法成功培养出人甲床上皮细胞,所得细胞纯度高并能传代培养,为深入研究甲床细胞的病理生理及分子生物学奠定了基础。
Objective: To investigate the method of in vitro culture and passage of nail bed epithelial cells. Methods: The experiment was performed in the Central Laboratory of First Hospital of Jilin University from October 2004 to April 2005. ① specimen disinfection, cut off the nail bed and cut. ② 2.5g / L trypsin + 3g / L ethylenediaminetetraacetic acid 1: 1 mixture, 37 ℃ digestion repeated three times to obtain a cell suspension. ③ centrifuged twice, washed cells, adding serum-free keratinocyte culture fluid into a cell suspension, counted after inoculation in 25mm flask. ④ 37 ℃, volume fraction of 0.05 of CO2, saturated humidity incubator culture, changing fluid every 2 days 1. ⑤ to monolayer cells covered nearly 80% of the bottom of culture when the passage. ⑥ third generation of cells test items: under inverted microscope to observe cell growth and shoot; cell count, draw the growth curve; mouse anti-human keratin 17 antibody immunocytochemistry. Results: ① Cell growth: After inoculated cells were observed by inverted microscopy for 12h, most cells adherent, about 11d, cell growth density reached 70% ~ 80% of the bottom of the bottle. ② Immunocytochemistry results: more than 70% of cultured cells stained positive for specific keratin 17 antibody. ③ growth curve and population doubling time: one or two days after inoculation decreased the number of cells, since the third day the number of cells increased rapidly to 9 ~ 12d reached its peak. Log doubling cell population doubling time was 4.46d. Conclusion: The human nail bed epithelial cells were successfully cultured by enzymatic digestion. The obtained cells were highly purified and subcultured, which lays the foundation for further study on the pathophysiology and molecular biology of nail bed cells.