兔气管纤毛上皮细胞原代培养的生物学特性及其体外生长规律  

Biological characters of tracheal ciliated epithelial cells of primary culture in rabbits and the growth pattern in vitro

作　　者：张龙芳[1] 崔鹏程[1] 陈文弦[1] 刘智[1] 李贵泽[1] 孙永柱[1] 

机构地区：[1]解放军第四军医大学唐都医院耳鼻咽喉科,陕西省西安市710038

出　　处：《中国临床康复》2006年第1期34-36,i0002,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金(30171007)~~

摘　　要：目的:建立兔气管纤毛上皮细胞原代培养模型,观察其体外培养条件下的生物学特性并确定其体外生长规律。方法:实验于2005-03/07在解放军第四军医大学唐都中心实验室完成。①纤毛上皮细胞提取及培养:日本大耳兔20只,利用酶消化法分离获取兔气管纤毛上皮细胞,无血清生长因子培养基(成分:DMEM:Ham’sF12为1∶1,并加入以下激素及生长因子:10mg/L胰岛素、5mg/L转铁蛋白、20μg/L甲状腺素、0.4mg/L氢化可的松、7.5mg/L内皮细胞生长支持物、25μg/L表皮生长因子、1mmol/L-谷氨酰胺,100IU/mL青霉素,50mg/L链霉素)体外培养。②细胞纯度及鉴定:用抗角蛋白单克隆抗体进行SP法免疫细胞化学反应,二氨基联苯胺显色阳性为上皮细胞。扫描电镜观察细胞表面结构。结果:①气管上皮细胞的分离:低温消化加刷洗法获得的气管上皮细酶胞数量较多,呈圆形,较大,悬浮旋转。②气管上皮细胞生长状态和形态特征:皿接种细胞24h后,贴壁率约为60%,细胞增大,并进入分裂套相。五六天后细胞增殖旺盛,部分无纤毛上皮细胞汇合呈多角形铺路石样相嵌生长,在相差显微镜下放大200～400倍清晰可见纤毛呈海葵样向心摆动活跃,第3周纤毛逐渐消失成遗迹,细胞凋亡。③纤毛细胞的电镜观察:见纤毛由细胞顶膜伸出,大部分集结成束,除纤毛外,还可有稠密的较细短的微绒毛由细胞顶膜伸出。④气道上皮细胞的免疫细胞化学鉴定:抗角蛋白单克隆抗体免疫组织化学染色阳性,细胞达(91±3)%。结论:酶消化法分离、无血清生长因子培养基培养,以建立兔气管纤毛可上皮细胞的原代培养模型,具有成为气管组织工程种子细胞的应用价值。AIM： To establish appropriate rabbit models of tracheal ciliated epithelial cells primary culture, and observe its bionomics and growth pattern during monolayer cultured in vitro. METHODS： The experiment was carried out in the central laboratory of Tangdu Hospital of the Fourth Military Medical University of Chinese PLA between March and July 2005. ①Extraction and culture of ciliated epithelial cells： Twenty Japanese white big-ear rabbits were used. Rabbit tracheal ciliated epithelial cells were obtained by means of enzyme digestion, and in vitro cultured with serum-free medium （DMEM： Ham＇s F12 was 1：1, following hormones and growth factors were supplemented： 10 mg/L insulin, 5 mg/L transferring, 20 μg/L thyroxin, 0.4 mg/L hydrocortisone, 7.5 mg/L endothelial growth supporter, 25 μg/L epidermal growth factor, 1 mmol/L glutamine, 100 IU/mL benzylpenieillin, 50 mg/L estreptomicina）. ②Cell purity and the evaluation： Anti-keratin monoclonal antibody was applied in the SP method immunohistochemical stain, positive stain of diaminobenzidine was epithelial cell. The scanning electron microscope （SEM） was employed to observe the structure of cell surface. RESULTS：①Isolation of tracheal epithelial cells： The tracheal epithelial cells obtained by means of enzyme hypothermia digestion plus washing were more in number, the shapes were round and bigger, suspended and rotated. ②Growth pattern and morphological characters of tracheal epithelial cells： At 24 hours after inoculation, the adherent rate was about 60%, the cells were enlarged and entered the splitting phase. The cell proliferation became high after 5 or 6 days, part of the without ciliated epithelial ceils confluent and showed multi-angle and pave-stone like grew. It was observed under phase contrast microscope of 200-400× that clear cilia actively swang centripetally like sea flower, and the cilia gradually disappeared at 3 weeks, and apoptosis could also be observed. ③Ciliated cells observed under electron microscope： I

关 键 词：细胞培养 纤毛上皮细胞 兔 原代培养 生物学特性 体外生长 

分 类 号：R318[医药卫生—生物医学工程]