小鼠气管平滑肌原代细胞的分离、培养与鉴定  

Isolation,culture and identification of primary airway smooth muscle cells from mice

作　　者：叶致豪 许文豪[1] 刘庆华[1] 沈金花[1] 彭勇波[1] 

机构地区：[1]中南民族大学生命科学学院医学生物研究所武陵山区特色资源植物种质保护与利用湖北省重点实验室,武汉430074

出　　处：《中国实验动物学报》2016年第4期364-368,共5页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金项目(30900816;31371307);湖北省自然科学基金项目(2014CFC116)

摘　　要：目的建立一种可靠的通过组织块贴壁法分离培养原代小鼠气管平滑肌细胞及免疫组化鉴定的方法。方法体视显微镜下立体分离小鼠气管平滑肌组织,组织块贴壁法培养原代细胞,对分离培养细胞通过免疫组化方法进行鉴定,并用MTT法对其增殖特性进行检测。结果从BALB/c雄性小鼠分离气管平滑肌组织,剪碎为1 mm3,用含1%青-链霉素的PBS及培养液漂洗,使组织块贴于培养皿底,并加入5 m L培养液,放入37℃、5%CO2的细胞培养箱培养,3~5 d后有明显梭状细胞从组织块爬出,5~6 d后,细胞可见明显＂峰-谷＂结构。经免疫荧光鉴定,在传代、纯化后,可得纯度为99%以上的气管平滑肌细胞。用MTT法测量其生长曲线。结论本方法操作简单、经济,获得的气管平滑肌细胞具有较好增殖能力,细胞数量和纯度能够满足后续细胞生物学实验研究的需要。Objective To develop a reliable method for the primary culture of airway smooth muscle cells（ ASMCs） from mice by adherent tissue culture and to identify them by immunofluorescence microscopy. Methods Airway smooth muscle（ ASM） tissue was isolated from BALB / c mice under dissecting microscope,and cut into 1 mm3 pieces.These tissue blocks were washed with PBS with 1% penicillin and streptomycin,and adhered on 6 cm culture dish with 5m L culture media. The dishes were incubated at 37℃ in an incubator with 5% CO2. The obtained primary ASMCs were identified by immunofluorescence microscopy,and the cell proliferation was measured by MTT assay. Results We observed that obvious fusiform cells grew out from tissue blocks within 3 to 5 days. After 5 to 6 days,＂hill-valley＂structure was observed. After cell passage and purification,the immunofluorescence microscopy showed that the purity of isolated ASMCs reached up to 99%. The growth curve of ASMCs was constructed by MTT assay. Conclusions Obtained ASMCs from this simple and economical method show a preferable proliferation ability,density and purity,and can satisfy the need for cell biology research.

关 键 词：气管平滑肌细胞 原代培养 组织块贴壁法 免疫荧光 MTT法 小鼠 

分 类 号：Q95-33[生物学—动物学]