胰酶分次消化及无血清角化细胞培养基培养甲床上皮细胞的体外实验

Nail bed epithelia in vitro cultured with the method of enzyme digestion by different times in serum-free medium containing keratinocytes

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。

AIM：To study the methods of culture and passage of nail bed epithelia in vitro. METHODS： The experiment was carried out in the central laboratory of the First ttospital of Jilin University between October 2004 and April 2005. ① Nail beds were minced into small pieces after disinfection of samples.②2.5 g/L trypsin and 3 g/L ethvlene dinitrilotetraaeetic acid was made into mixture of 1：1, and then digested for 3 times at 37℃ to obtain cell suspension. ③ After eentrifugation for twice, the cells were washed, and serum-free medium containing keratinocytes was added to make cell suspension, and then they were inoculated into the 25 mm culture bottle after counting. ④ The cells were cultured at 37℃ in the incubator containing CO, with volume fraction of 0.05 and saturate humidity, and the liquid was changed once every, two days. ⑤ They were passaged till monolayer cells spread to more than 80% of the bottom of the culture bottle. ⑥ Detected items of cells of the 3rd passage： The growth of the cells was observed under inverted microscope and photographed, the cells were counted, and the growth curve was drawn, the rat-anti-body keratin 17 antibody was used in the immunohistochemistry. RESULTS： ① Cell growth： Under inverted microscope, the most of the cells adhered at 12 hours after inoculation, the cell growth density at about 11 days reached 70%-80% of the bottom of bottle, and they arranged like cobbles. ② Results of immunohistochemistry： More than 70% of the cells were positively stained with the monoclonal antibody （K17）, which was expressed in nail bed and matrix in vivo, indicating that the cells oringinated from the nail bed. ③ The number of cells decreased within 1-2 days after inoculation, and then the number of cells increased rapidly from the 3rd day, and reached the peak at 9-12 days, the doubling time of cells at the logarithm growth period was 4.46 days. CONCLUSION： Nail cells could be succesfully euhured by means of enzyme digestion, and the purity of the obtained cells was high and they can be passaged, which lays basis for the further study of the pathophysiology and molecular biology of nail bed cells.