CAJ | 학술논문

背景：人类原代肺脏上皮细胞在体外难以分离培养，表现为组织来源有限、细胞存活率低、增殖速度慢，缺乏上皮细胞表型分化能力等。 　　目的：体外扩增人细支气管上皮细胞，建立其气液相分化模型，用于肺脏上皮细胞功能研究。 　　方法：采用Pronase和DnaseⅠ联合消化法分离人细支气管上皮细胞。利用ROCK激酶抑制剂培养体系对其进行扩增，免疫荧光染色鉴定细胞类型。建立气液相培养模型，扫描电镜、相差显微镜和免疫荧光染色鉴定细胞分化类型。 　　结果与结论：通过 ROCK 激酶抑制剂培养体系，体外成功培养扩增了人细支气管上皮细胞。扩增细胞经免疫荧光染色鉴定绝大部分都表达基底细胞标记角质蛋白14，提示人细支气管的基底细胞可能是肺脏上皮干细胞的主要亚群。同时，扩增细胞在气液相培养条件下分化成纤毛细胞和无纤毛柱状细胞，表明ROCK激酶抑制剂培养体系扩增的上皮细胞仍然保持干细胞的增殖分化能力，体外气液相培养模型可以促进人细支气管上皮细胞分化。
배경：인류원대폐장상피세포재체외난이분리배양，표현위조직래원유한、세포존활솔저、증식속도만，결핍상피세포표형분화능력등。 　　목적：체외확증인세지기관상피세포，건립기기액상분화모형，용우폐장상피세포공능연구。 　　방법：채용Pronase화DnaseⅠ연합소화법분리인세지기관상피세포。이용ROCK격매억제제배양체계대기진행확증，면역형광염색감정세포류형。건립기액상배양모형，소묘전경、상차현미경화면역형광염색감정세포분화류형。 　　결과여결론：통과 ROCK 격매억제제배양체계，체외성공배양확증료인세지기관상피세포。확증세포경면역형광염색감정절대부분도표체기저세포표기각질단백14，제시인세지기관적기저세포가능시폐장상피간세포적주요아군。동시，확증세포재기액상배양조건하분화성섬모세포화무섬모주상세포，표명ROCK격매억제제배양체계확증적상피세포잉연보지간세포적증식분화능력，체외기액상배양모형가이촉진인세지기관상피세포분화。
BACKGROUND:Primary human lung epithelial cel s are difficult to be isolated and cultured in vitro, which is characterized as limited sources, low cel viability, slow proliferation capacity, and lacking of differentiation capability. OBJECTIVE:To establish an air-liquid interface model of lung epithelium by in vitro proliferation of human bronchiolar epithelial cel s, which is used for research on function of lung epithelial cel s. METHODS:Primary human bronchiolar epithelial cel s were isolated using Pronase and DNase I combined digestive methods, and then proliferated using medium containing ROCK kinase inhibitor. The proliferated cel s were used for establishment of the air-liquid interface epithelium model. Cel differentiation was identified using scanning electron microscope, phase contrast microscope and immunofluorescent staining. RESULTS AND CONCLUSION:The primary human bronchiolar epithelial cel s could be expanded successful y using medium containing ROCK kinase inhibitor, and the basal cel marker Cytokeratin14 was preferential y expressed in the proliferated cel population, indicating that these basal cel s might be the main subpopulation of human lung epithelial stem cel s. Subsequently, the proliferated cel s under the air-liquid interface could differentiate into ciliated cel s and non-ciliated column cel s. The results suggest that the proliferation and differentiation of human bronchiolar epithelial cel s were maintained in the presence of ROCK kinase inhibitor, and the air-liquid interface could promote the differentiation of human bronchiolar epithelial cel s.