一种高效的小鼠肺单细胞悬液制备方法的研究

A method for isolation of single-cell suspensions from lung in mice

目的:应用胶原酶Ⅴ酶消化法建立一种高效的小鼠肺单细胞悬液制备方法。方法:小鼠处死后取出肺脏,将其剪碎,应用胶原酶Ⅴ(175 U.ml-1)消化,37℃孵育1 h,随后进行细胞计数、活细胞百分率和流式细胞仪检测。结果:(1)光镜下见胶原酶Ⅴ消化所得小鼠肺细胞多种多样,大小不一,细胞形态完整,分散度好;(2)30 mg小鼠肺组织可提取细胞数量为(1～3)×106个,肺单细胞悬液中活细胞百分率均在95%以上;(3)流式细胞术检测可见大量的肺细胞。结论:应用胶原酶Ⅴ酶消化法制备小鼠肺单细胞悬液高效、简便,为肺细胞生物学及肺部疾病发病机制研究提供了有效的肺单细胞制备方法。

Objective：To set up an effective method for isolation of single-cell suspensions from lung in mice.Methods：Isolation of lung cells was performed by type Ⅴ collagenase digestion.Briefly,lungs were finely dissected into small pieces using scissors and then digested with type Ⅴ collagenase（175 U·ml-1）for 1 h at 37℃.Then,single-cell suspensions were detected by cell counts and flow cytometry.Results：（1） By type Ⅴ collagenase digestion,higher single cell output rate,more intact cells,little cell fragments and little cell conglomeration were found in lung cell suspension.（2） It can be extracted（1-3）× 106 cells from 30 mg lung tissue in mice,and the percentage of living cells in lung single-cell suspension was 95% or more.（3） Flow cytometry showed a large number of lung single cells.Conclusion：The enzymatic digestion method by type Ⅴ collagenase is a simple,effective,stable and reliable method for the preparation of lung single-cell suspensions,which could be widely used to isolate lung tissues for in vitro functional tests.