中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (15): 2761-2764.doi: 10.3969/j.issn.1673-8225.2010.15.024

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

构建原代分离培养与鉴定小鼠肺泡Ⅱ型上皮细胞的方法及模型

郑金旭，黄振杰，汤  艳，丁  明   

  1. 江苏大学附属医院呼吸科，江苏省镇江市  212001
  • 出版日期:2010-04-09 发布日期:2010-04-09
  • 作者简介:郑金旭☆，男，1961年生，江苏省宜兴市人，1994年上海医科大学(现复旦大学上海医学院)毕业，博士，教授，博士生导师，主任医师，主要从事肺间质病与肺癌的研究。 jxuzh135@163.com
  • 基金资助:

    课题受卫生部科研项目(wkj2006-2-026)，江苏省“333工程”资助项目(苏人才办2007-16-09)资助。

Isolation, primary culture and identification of type Ⅱ alveolar epithelial cells from mice  

Zheng Jin-xu, Huang Zhen-jie, Tang Yan, Ding Ming   

  1. Department of Respiratory Medicine, Affiliated Hospital of Jangsu University, Zhenjiang  212001, Jiangsu Province, China
  • Online:2010-04-09 Published:2010-04-09
  • About author:Zheng Jin-xu☆, Doctor, Professor, Doctoral supervisor, Chief physician, Department of Respiratory Medicine, Affiliated Hospital of Jangsu University, Zhenjiang 212001, Jiangsu Province, China jxuzh135@163.com
  • Supported by:

    the Science Research Foundation of Ministry of Health, No. wkj2006-0-026*; “333” Project of Jiangsu Province, No. 2007-16-09*

PDF

743

被引次数

21

摘要：

背景：肺泡Ⅱ型上皮细胞在肺发育和肺功能调节中起重要作用，它与肺纤维化和肺癌等疾病的发生发展有密切联系，为了对这些疾病进行体外实验研究，必须对肺泡Ⅱ型上皮细胞进行分离、原代培养与鉴定以建立一个稳定高效的细胞模型。
目的：建立一套可靠的小鼠肺泡Ⅱ型上皮细胞分离、纯化、原代培养与鉴定的方法，为肺纤维化及肺癌等疾病的进一步研究奠定基础。
方法： ①用低浓度胰酶联合Ⅰ型胶原酶消化法，分离小鼠肺组织细胞成分。取出小鼠肺组织，剪成小块,加入胰蛋白酶，移出细胞悬液，加入含胎牛血清的DMEM中止消化；同法用胰蛋白酶再消化1次，中止消化后加入Ⅰ型胶原酶，再中止消化。②经差速离心差速贴壁和免疫黏附法纯化肺泡Ⅱ型上皮细胞，进行原代培养。将肺细胞悬液接种入包被小鼠IgG的培养皿中培养，吸出含未黏附细胞的液体接种于另一包被小鼠IgG的培养皿中培养，吸出未黏附细胞，去上清，重悬沉淀，将细胞接种于培养皿和培养板中培养。倒置显微镜观察细胞形态及生长特点，测定肺泡Ⅱ型上皮细胞产量、纯度及活力，免疫细胞化学染色鉴定肺泡表面活性蛋白A和肺泡表面活性蛋白C的表达，透射电镜鉴定肺泡Ⅱ型上皮细胞特异性细胞结构。
结果与结论：用此方法，每只小鼠获得肺泡Ⅱ型上皮细胞(4.8±1.2)×106，纯度达到(85.0±2.4)%，细胞活力为(92.0±2.4)%，倒置显微镜下原代肺泡Ⅱ型上皮细胞呈圆形或立方形，细胞质内有大量反差明显的细小颗粒，细胞为岛状生长方式；免疫细胞化学鉴定，肺泡表面活性蛋白A呈棕黄色，肺泡表面活性蛋白C呈绿色，均定位表达于细胞浆；透射电镜见特征性板层小体和细胞游离面大量微绒毛。证实利用胰酶联合胶原酶消化，差速离心差速贴壁和免疫黏附纯化的方法可获得高产量高纯度的AECⅡ，能满足一般的体外实验要求。

关键词: 小鼠, 肺泡Ⅱ型上皮细胞, 原代培养, 分离, 鉴定, 心肺组织工程

Abstract:

BACKGROUND: Type Ⅱ alveolar epithelial cells (AECⅡ) play an important role in lung development and regulation of lung function. It is closely related with the genesis and development of pulmonary fibrosis, lung cancer and so on. In order to make an in vitro study of these diseases, a stable and effective cell model for isolation, primary culture and identification of AECⅡ should be established.
OBJECTIVE: To develop a reliable method for the isolation, purification, primary culture and identification of AEC Ⅱ from mice, and provide a foundation for the further study of pulmonary fibrosis and lung cancer.
METHODS:  ①The lung tissue of mice was digested with low-concentration trypsin and collagenase Ⅰ. That is, lung tissues of mice were cut into small pieces, digested with trypsin, and terminated the digestion by adding DMEM containing fetal bovine serum. The cells were digested once again using trypsin, and adding collagenase Ⅰ after termination. ②AEC Ⅱ was purified by differential centrifugation and immune adherence for primary culture. The pneumocytes suspension was incubated in IgG culture plate, and liquid containing non-adherent cells were once again cultured in IgG culture plate, then supernatant were abandoned, and the cells were cultured in culture dish and culture plate. The morphology and growth characteristics of primarily cultured AEC Ⅱ were observed by an inverted microscope; the yield, purity and viability of AEC Ⅱ were determined. AEC Ⅱ was identified by immunocytochemical staining surfactant apoprotein A (SP-A) and SP-C, and the specific cell structure was observed under a transmission electron microscope.
RESULTS AND CONCLUSION: By this method, an amount of (4.8±1.2)×106 AEC Ⅱ were harvested from each mouse with a purity of (85±2.4)% and a viability of (92±2.4)%. Under an inverted microscope, AEC Ⅱ in primary culture showed a shape of round or cube and an island-like growth. The immunocytochemistry showed that the cytoplasm presented buffy SP-A and green SP-C. Typical features of AEC Ⅱ including lamellar bodies and microvilli could be seen under a transmission electron microscope. The results demonstrated that, AECⅡ of great amount and high purity could be obtained by this method, which meets the requirement of a cell model establishing for further in vitro study.

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