小鼠Lewis肺癌细胞非对称分裂细胞系的建立及其干性特征

Estabilshment and stemness identification of an asymmetric dividing cell line derived from mouse Lewis lung cancer cells

目的从小鼠Lewis肺癌细胞中分离建立非对称分裂细胞系,并初步鉴定其干性特征,为研究非对称分裂在癌症生物学中的作用奠定基础。方法通过8次连续悬浮聚球,随后5次单细胞克隆的方法,从小鼠Lewis肺癌细胞亲代细胞(LLC-parental cells)中得到非对称分裂细胞系(LLC-ASD cells);通过Brdu标记的免疫荧光实验检测LLC-ASD cells中细胞非对称分裂;进行RT-qPCR、克隆斑成斑实验、单细胞琼脂球形成实验、单细胞克隆形成实验对比两细胞系干性特征;并通过同种小鼠肺部移植成瘤实验和裸鼠皮下成瘤实验分别对比两种细胞系体内转移和成瘤能力。结果 LLC-ASD cells的非对称分裂比例达50%。LLC-ASD cells的克隆斑、悬浮球形成数量和单细胞克隆形成率均分别高于LLC-parental cells(P<0.05),LLC-ASD cells原位种植转移能力较LLC-parental cells明显增强,LLC-ASD cells的裸鼠皮下接种致瘤能力也高于LLC-parental cells(P<0.05)。结论成功建立了小鼠Lewis肺癌细胞非对称分裂细胞系(LLC-ASD cells),并且它表现出了一定干性特征,为研究非对称分裂在肿瘤中的作用奠定了基础。

Objective To establish an asymmetric dividing cell line（ LLC-ASD cells） derived from mouse Lewis lung carcinoma cancer cells（ LLC-parental cells）,and to investigate its stemness features in order to lay a foundation for depth studying the function of asymmetric dividing in the cancer biology. Methods In order to obtain asymmetrically dividing LLC cells（ LLC-ASD cells） derived from LLC-Parental cells,8 times of consecutive culture,enrichment and collection of floating spheriod forming cells followed by 5 times of consecutive single cell cloning were conducted. Immunofluorescence assay was used to visualize and quantify the rate of asymmetric division in LLC-ASD cells labeled by Brd U. For comparing the stem characteristics of LLC-Parental and LLC-ASD,RTqPCR,clonogenic assay in 6-well plate,single cell spheroid formation assay with agar in 6-well plate and 96-wellplate single cell cloning assay were conducted. In vivo,LLC-parental cells and LLC-ASD cells were subcutaneouslytransplanted in nude mice to determine the effect of the difference in stem cell like properties on tumorigeneicy. The same lung transplantation into tumor experiment in mice were used to compare the differences in cancer biology.Results Asymmetric dividing cells were found in LLC-ASD cell culture through the Brd U immunofluorescence assay and the rate of asymmetric division in the anaphase cells was as high as 50%.According to the clonogenic assay in 6-well plate,single cell and spheroid formation assay with agar in 6-well plate and 96-well-plate single cell cloning assay in LLC-ASD cells,the results showed that they were more prominent than those in the LLC-Parental cells（ P〈0. 05）. In vivo,the tumor metastasis potentials of LLC-ASD was enhanced than that of LLC-parental when transplanted to the C57 mice. Further,the tumorigenic potentials of LLC-ASD cells was also increased.Conclusions The asymmetric dividing cell line derived from mouse Lewis lung carcinoma cancer cell line（ LLC-ASD cells） is established which exhibits stemness properties. The establishment and characterization of this model will facilitate the research on the function of asymmetric cell dividing in cancer biology.