改良组织块贴壁法培养小鼠气道平滑肌细胞及鉴定  

Improvement and identification of tissue⁃piece inoculation mouse airway smooth muscle cells

作　　者：赵爱玲 姚冬 ZHAO Ailing;YAO Dong(Department of Respiratory&Critical Care Medicine,The Second Affiliated Hospital of Guilin Medical University,Guilin 541000,Guangxi,China;The Key Laboratory of Respiratory Diseases,Education Department of Guangxi Zhuang Autonomous Region/Guangxi Medical and Health Key Discipline Construction Project,Guilin 541000,Guangxi,China)

机构地区：[1]桂林医学院第二附属医院呼吸与危重症医学科,桂林541000 [2]广西高校呼吸疾病重点实验室/广西医疗卫生重点学科,桂林541000

出　　处：《医学研究与战创伤救治》2023年第3期225-230,共6页Journal of Medical Research & Combat Trauma Care

基　　金：呼吸疾病国家重点实验室开放课题(SKLRD⁃OP⁃202110);广西研究生教育创新计划项目(YCSW2021249)。

摘　　要：目的采用改良组织块贴壁法建立小鼠气道平滑肌细胞体外培养模型。方法用I型胶原酶消化小鼠气道平滑肌组织块后再贴壁,培养出的小鼠原代气道平滑肌细胞经过细胞免疫荧光技术鉴定。结果相比单纯组织块贴壁法,改良组织块贴壁法获得的原代平滑肌细胞的纯度更高[(93.0%±1.8%)vs(96.1%±1.8%),P<0.01],酶消化法获得的平滑肌细胞的纯度(90.8%±1.9%)最低,与改良组织块贴壁法相比,差异具有统计学意义(P<0.01)。结果还显示P5代细胞与P1代细胞相比,平滑肌细胞生长形态较前改变,呈不规则形,且α⁃SMA阳性表达减弱,Vimentin阳性表达增强,标志着平滑肌细胞的纯度会随传代次数增加而逐渐降低。酶消化法获得的ASMCs的增殖速度较单纯组织块贴壁法和改良组织块贴壁法获得的ASMCs的增殖速度慢(P<0.01)。结论改良组织块贴壁法经济稳定,可在短期内培养出数量多、纯度高、活性好的小鼠气道平滑肌细胞,此法成功建立了体外培养小鼠气道平滑肌细胞的模型。Objective The in vitro culture of airway smooth muscle cells(ASMCs)provides an effective platform for the study of respiratory diseases,but the in vitro culture technology of mouse airway smooth muscle cells needs to be further improved.In this paper,the modified tissue adhesion assay was used to isolate and culture mouse airway smooth muscle cells in vitro.Methods Mouse airway smooth muscle tissue pieces were digested with type I collagenase and then attached to the bottom of cell culture flasks.The cultured mouse primary ASMCs were identified by the cell immunofluorescence staining.Results The purity of primary smooth muscle cells obtained by modified tissue⁃adhesionmethod was higher than that obtained by simple tissue⁃adhesion method[(93.0%±1.8%)vs(96.1%±1.8%),P<0.01],the purity of the cells obtained by the enzyme⁃digestion method was the lowest(90.8%±1.9%),and the difference was statistically significant compared with the modified tissue⁃adhesion method(P<0.01).The results also showed that the growth morphology of smooth muscle cells in P5 generation changed with more irregular than that in P1 generation,andα⁃SMA positive expression decreased and the positive expression of Vimentin increased,indicating that the purity of smooth muscle cells would gradually reduce with the increase of passage times.The proliferation rate of ASMCs obtained by enzyme⁃digestion method was slower than that obtained by simple tissue⁃adhesion method and modified tissue⁃adhesion method(P<0.01).Conclusion The improved tissue piece inoculation method is economical and stable and can cultivate mouse ASMCs with a large number,high purity,and good activity in a short period of time.This method has successfully established a model for culturing mouse ASMCs in vitro.

关 键 词：气道平滑肌细胞 小鼠 原代培养 

分 类 号：R526[医药卫生—内科学] R36[医药卫生—临床医学]