中国组织工程研究

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (28): 4582-4587.doi: 10.3969/j.issn.2095-4344.2015.28.028

• 干细胞移植 stem cell transplantation • 上一篇    下一篇

人细支气管上皮细胞的ROCK激酶抑制剂培养体系扩增及气液相模型的建立

贾元元1，2，何进喜3，孙颖飞1，2，韩 飞3，杨佳丽3，李 勇1，2，刘晓明1，2，3   

  1. 1西部生物资源保护与利用教育部重点实验室，宁夏回族自治区银川市 750021；
    2宁夏大学生命科学学院，宁夏回族自治区银川市  750021；
    3宁夏医科大学总医院，宁夏回族自治区银川市  750004
  • 出版日期:2015-07-02 发布日期:2015-07-02
  • 通讯作者: 刘晓明，博士，教授，博士生导师，西部生物资源保护与利用教育部重点实验室，宁夏回族自治区银川市 750021；宁夏大学生命科学学院，宁夏回族自治区银川市 750021；宁夏医科大学总医院，宁夏回族自治区银川市 750004
  • 作者简介:贾元元，女，1990年生，宁夏回族自治区石嘴山市人，汉族，宁夏大学生命科学学院在读硕士，主要从事肺脏上皮细胞的分离培养和生物学特性研究。
  • 基金资助:

    国家自然科学基金(31172278)

An air-liquid interface model of human lung epithelium generated from bronchiolar epithelial cells proliferated using medium containing ROCK kinase inhibitor 

Jia Yuan-yuan1, 2, He Jin-xi3, Sun Ying-fei1, 2, Han Fei3, Yang Jia-li3, Li Yong1, 2, Liu Xiao-ming1, 2, 3   

  1. 1Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western China, Yinchuan 750021, Ningxia Hui Autonomous Region, China;
     2College of Life Science, Ningxia University, Yinchuan 750021, Ningxia Hui Autonomous Region, China; 
    3General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Online:2015-07-02 Published:2015-07-02
  • Contact: Liu Xiao-ming, M.D., Professor, Doctoral supervisor, Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western China, Yinchuan 750021, Ningxia Hui Autonomous Region, China; College of Life Science, Ningxia University, Yinchuan 750021, Ningxia Hui Autonomous Region, China; General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Jia Yuan-yuan, Studying for master’s degree, Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western China, Yinchuan 750021, Ningxia Hui Autonomous Region, China; College of Life Science, Ningxia University, Yinchuan 750021, Ningxia Hui Autonomous Region, China
  • Supported by:

     the National Natural Science Foundation of China, No. 31172278

PDF

233

摘要：

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。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 干细胞, 培养, 人细支气管上皮细胞, 肺脏, ROCK激酶抑制剂, 气液相培养, 国家自然科学

Abstract:

BACKGROUND: Primary human lung epithelial cells are difficult to be isolated and cultured in vitro, which is characterized as limited sources, low cell 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 cells, which is used for research on function of lung epithelial cells.
METHODS: Primary human bronchiolar epithelial cells were isolated using Pronase and DNase I combined digestive methods, and then proliferated using medium containing ROCK kinase inhibitor. The proliferated cells were used for establishment of the air-liquid interface epithelium model. Cell differentiation was identified using scanning electron microscope, phase contrast microscope and immunofluorescent staining.
RESULTS AND CONCLUSION: The primary human bronchiolar epithelial cells could be expanded successfully using medium containing ROCK kinase inhibitor, and the basal cell marker Cytokeratin14 was preferentially expressed in the proliferated cell population, indicating that these basal cells might be the main subpopulation of human lung epithelial stem cells. Subsequently, the proliferated cells under the air-liquid interface could differentiate into ciliated cells and non-ciliated column cells. The results suggest that the proliferation and differentiation of human bronchiolar epithelial cells were maintained in the presence of ROCK kinase inhibitor, 
and the air-liquid interface could promote the differentiation of human bronchiolar epithelial cells. 
BACKGROUND: Primary human lung epithelial cells are difficult to be isolated and cultured in vitro, which is characterized as limited sources, low cell 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 cells, which is used for research on function of lung epithelial cells.
METHODS: Primary human bronchiolar epithelial cells were isolated using Pronase and DNase I combined digestive methods, and then proliferated using medium containing ROCK kinase inhibitor. The proliferated cells were used for establishment of the air-liquid interface epithelium model. Cell differentiation was identified using scanning electron microscope, phase contrast microscope and immunofluorescent staining.
RESULTS AND CONCLUSION: The primary human bronchiolar epithelial cells could be expanded successfully using medium containing ROCK kinase inhibitor, and the basal cell marker Cytokeratin14 was preferentially expressed in the proliferated cell population, indicating that these basal cells might be the main subpopulation of human lung epithelial stem cells. Subsequently, the proliferated cells under the air-liquid interface could differentiate into ciliated cells and non-ciliated column cells. The results suggest that the proliferation and differentiation of human bronchiolar epithelial cells were maintained in the presence of ROCK kinase inhibitor, 
and the air-liquid interface could promote the differentiation of human bronchiolar epithelial cells. 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Lung, Bronchioles, Epithelial Cells

中图分类号: