腺样囊性癌细胞系中肿瘤干细胞的生物学特性初探

Cancer stem cell:seeking its evidences in adenoid cystic carcinoma cell line

目的：对腺样囊性癌细胞系ACC-2中不同表型的肿瘤细胞生物学特性进行分析和研究,为肿瘤干细胞学说提供实验依据。方法：采用单细胞体外培养法,了解腺样囊性癌细胞(ACC-2)的分裂、增殖特点；采用免疫组化方法,检测ACC-2细胞表面CD44和CD24表达的差异性；应用免疫磁珠技术,分离不同表型的ACC-2细胞后,以裸鼠成瘤试验了解不同表型ACC-2细胞的成瘤能力。采用SPSS10．0统计软件包对所得数据进行X^2检验。结果：体外单细胞培养的ACC-2,仅有4．5%可持续分裂、增殖。CD44-和CD44+/CD24+细胞在体外培养条件下均无长期存活能力。不同表型的ACC-2动物成瘤试验结果显示,CD44+/CD24-亚群的ACC-2细胞最低成瘤接种细胞数显著低于常规ACC-2细胞,CD44-和CD44+/CD24+细胞则均无成瘤能力。免疫组化染色证实,CD44+/CD24-肿瘤细胞具有分化为其他表型肿瘤细胞的能力。结论：细胞表面抗原为CD44+CD24-的肿瘤细胞仅占ACC-2肿瘤细胞的极少部分,这些细胞具有极强的增殖能力和分化为其他表型肿瘤细胞的能力。ACC-2肿瘤细胞的增殖和成瘤能力源于CD44+/ CD24-细胞亚群。未分离的ACC-2细胞与CD44+/CD24-ACC-2细胞在与细胞分化、凋亡、黏连、信号传导等有关的基因上存在差异。提示ACC-2中存在肿瘤干细胞,而CD44+/CD24-是ACC-2肿瘤干细胞必须兼备的表面标志。

PURPOSE： The present study is designed to seek the evidences of tumor stem cell and reveal its impact on tumorigenesis of ACC-2 cell line by analyzing the biologic characteristics of different sub-clones of adenoid cystic carcinoma cell line （ACC-2）. METHODS： In vitro individual cell culture was employed to observe the proliferating character of ACC-2 cells. The expression of CD44 and CD24 of ACC-2 cells were investigated by inmmnohistochemical assay. Immunomagnetic isolation of different phenotypes of ACC-2 cells, followed by cell culture, was used to study the proliferating abilities of different clusters of the cell line. The hetero-transplanted tumor mold was established using BALB/C nude mice by subcutaneous injection of tumor cells. The tumorigenic properties of the different clusters were investigated in vivo. RESULTS： The result of in vitro individual cell culture revealed that only 4.49% of cultured ACC-2 cell underwent division, proliferation and cell cloning, which indicated preservation of the prolonged live ability. All CD44- and CD44 ＋/CD24 ＋ cells were failed to be eternal alive in the condition of in vitro individual cell culture. According to the results of in vivo tumorigenic study, the minimal cell quantity to develop a subcutaneous transplanted tumor by CD44＋/CD24-cells was less than that of non-isolated ACC-2 cells and CD44＋ cells. The CD44-and CD44＋/ CD24＋ were failed to develope transplanted tumors. Immunohistochemical study proved CD44＋/CD24- ACC-2 cell could differentiate into cells of other phenotypes. CONCLUSIONS： It was suggested that heterogeneity exists in ACC-2 cell lines. There are some ACC-2 cells which display the following properties：They consisted in a very small portion of all ACC-2 cells. They were possessed of remarkable proliferating ability, and represented the sole cluster that can establish cell clone and alive eternally. They borne special phenotypes, and CD44＋/CD24- were necessary. During the proliferation and differentiation, the expressing fashion of CD44 and CD24 might be changed. The tumorigenic ability of CD44＋/CD24- cells were stronger than CD44＋ and non-isolated ACC-2 cells. Eliminating of this cluster from ACC-2 would actually deprive the tumorigenic ability of the cell line. Supported by National Natural Science Foundation of China （Grant No. 30271425）.