三种方法培养慢性阻塞性肺疾病大鼠气道平滑肌细胞的比较

背景：原代培养方法被广泛应用于正常和哮喘大鼠气道平滑肌细胞的培养,但少见用于慢性阻塞性肺疾病大鼠气道平滑肌细胞培养的报道。目的：建立大鼠慢性阻塞性肺疾病模型,比较组织块法、酶消化法和改良组织块消化法原代培养模型大鼠气道平滑肌细胞生长情况的差异。方法：将16只清洁级雄性健康SD大鼠随机分成2组,对照组正常饲养,慢性阻塞性肺疾病组用熏烟法建立大鼠慢性阻塞性肺疾病模型,显微镜下观察大鼠肺组织病理学特点。分别应用上述3种方法原代培养慢性阻塞性肺疾病大鼠气道平滑肌细胞,相差显微镜下观察细胞形态,用α-平滑肌肌动蛋白免疫组织化学染色鉴定细胞类型。结果与结论：经病理学证实成功构建慢性阻塞性肺疾病大鼠模型。在倒置相差显微镜下见培养的细胞表现为典型的＂谷-峰状＂生长。经免疫细胞化学染色后,可见胞质呈棕色阳性反应,所培养的细胞有94%以上为气道平滑肌细胞。3种方法在耗时和细胞质量方面均无明显差别,但组织块法更经济、稳定可靠和简单。

Comparison of three methods for culturing the air smooth muscle cells of rats with chronic obstructive pulmonary disease

BACKGROUND：Methods of primary culture have been extensively used to culture airway smooth muscle cells in normal or asthma rats,but the same way to culture airway smooth muscle cells in chronic obstructive pulmonary disease rats has rarely been reported.OBJECTIVE：To establish the rat chronic obstructive pulmonary disease model,and to compare the different growth conditions of airway smooth muscle cells in rat chronic obstructive pulmonary disease models by three methods：attachment-block culture and enzymatic dispersion as well as improved tissue-piece digestion inoculation.METHODS：Sixteen healthy male clean-grade Sprague-Dawley rats were randomly divided into chronic obstructive pulmonary disease group and control group with eight rats in each group.Rats in the control group were fed normally.The rat chronic obstructive pulmonary disease models were established in the chronic obstructive pulmonary disease group by cigarette smoke exposure.The pathologic characteristics of lung tissues of the rat models were observed under microscope.The airway smooth muscle cells of rat chronic obstructive pulmonary disease model were cultured from primary generation using the three ways mentioned above,respectively.The morphology of the cultured cells was observed under phase contrast microscope and α-smooth muscle actin immunostaining was used to identify the cell types.RESULTS AND CONCLUSION：The rat chronic obstructive pulmonary disease models were established successfully,which was proved by pathology.The cultured cells demonstrated the typical ＂hill and valley＂ appearance under phase contrast microscope.After α-smooth muscle actin immunostaining,brown positive reaction was observed in the cytoplasm.There were over 94% cultured cells identified to be airway smooth muscle cells.There was no significant difference of consuming time and quality of cells in these three ways.Compared with the other two methods,attachment-block culture was a more economical and stable and less complicated way to culture airway smooth muscle cells in rat chronic obstructive pulmonary disease models.