改良组织贴壁法分离培养新生乳小鼠原代肺成纤维细胞的研究  

Study on Isolation and Culture of Primary Lung Fibroblasts from Newborn Mice by Improved Tissue Adhesion Method

作　　者：郑晓丹 王婷[1] 胡玉海[1] ZHENG Xiaodan;WANG Ting;HU Yuhai(Department of Clinical Laboratory,Wuhan Hankou Hospital,Wuhan 430010,China)

机构地区：[1]武汉市汉口医院检验科,武汉430010

出　　处：《现代检验医学杂志》2024年第6期206-210,共5页Journal of Modern Laboratory Medicine

基　　金：武汉市卫健委中医药科研项目面上项目(立项编号:WZ24A25)。

摘　　要：目的建立改良版简单高效的原代小鼠肺成纤维细胞(lung fibroblast,LFB)分离培养方案,研究其体外生长特性。方法无菌条件下,分别取3天龄小鼠和8周龄小鼠肺组织,剪成1mm3大小,分别采用含10g/dl胎牛血清(fetal bovine serum,FBS)和20g/dl胎牛血清的高糖DMEM培养液进行组织贴壁法培养。采用差时贴壁法对LFB进行纯化,倒置显微镜下动态观察细胞生长状态及贴壁状态,采用流式细胞特异性分子染色法对LFB进行鉴定,传代培养后的第3代细胞采用CCK-8(cell counting kit-8)法检测细胞活性。结果3天龄小鼠在20g/dl FBS浓度条件下采用改良组织贴壁法进行培养,培养后第2天开始向周边呈放射状长出细胞,第7天细胞生长密度达90%,细胞形态呈长梭形。经差时贴壁法纯化后,CD140a阳性且CD45阴性细胞占比可达90%以上,连续传代3次后保持较好细胞活性。结论改良的组织贴壁法可简单高效地获得大量纯化的、活性好的小鼠LFB,为肺部炎症、肿瘤及药物体外疗效等研究奠定基础。Objective To establish an improved simple and efficient method for isolation and culture of primary lungfibroblasts(LFB)from neonatal mice,and study their growth characteristics in vitro.Methods The lungs of 3-days-old mice or 8-weeks-3 old mice were taken under sterile conditions,and the stromal tissue was cut into 1mm tissue mass.High-glucose DMEM culture medium containing 10g/dl fetal bovine serum(FBS)or 20g/dl FBS was used for tissue adhesion culture.The LFB was purified by the differential time adhesion method,and the growth morphology and adherence state of the cells were observed dynamically under an inverted microscope.The primary LFB was identified byflow cytometry.The activity of the third-generation cell after culture was detected by CCK-8 assay.Results Lung tissues in mice at 3 days of age and 20g/dl FBS concentration cultured by the improved tissue mass adherent cultured method began to grow radially to the periphery on the 2nd day.On the day 7th,the cells growth density reached 90%,and the cell morphology was as a spindle.The CD140a positive and CD45 negative cells reached more than 90%after purification by differential time adhesion method,and the cells still maintain good cell activity after cultured for 3 generations.Conclusion The improved tissue adhesion method can obtain a large number of purified mice LFB with good activity simply and efficiently,which lays a foundation for the study of lung inflammation,tumors and in vitro efficacy of drugs.

关 键 词：肺成纤维细胞 原代培养 组织贴壁法 流式细胞染色 

分 类 号：R-332[医药卫生]