人泪腺腺样囊性癌细胞系LACC中肿瘤干细胞的分离、培养和鉴定

Isolation and identification of cancer stem cells from human LACC cell line

目的 研究从人泪腺腺样囊性癌细胞系LACC中分离培养泪腺腺样囊性癌干细胞的方法,并对其特性进行鉴定.方法 实验研究.将LACC细胞消化、离心制成单细胞悬液,悬浮于无血清培养基（SFM）中获得肿瘤细胞微球体;应用有限稀释和单克隆形成实验确定该细胞系中肿瘤干细胞的比例;MTT法检测肿瘤微球体的增殖能力以及其耐药性;流式细胞仪检测细胞表面分子标志CD44＋/CD24-在两种细胞中的表达情况;向SFM中滴加含有血清培养基诱导LACC肿瘤微球体分化,并观察其形态学变化.细胞增殖实验中两种细胞的吸光度（A）值和两种细胞的IC50比较采用t检验.结果 LACC细胞系中有（0.92±0.02）％的肿瘤细胞在无血清培养基中能够存活增殖形成自由漂浮的细胞球并可稳定传代;LACC肿瘤微球体和LACC贴壁细胞的IC50分别是22.53 mg/L,11.06mg/L（t =37.94,P ＜0.05）,说明肿瘤微球体对顺铂具有更强的耐药性.流式细胞仪表面标志检测,LACC细胞CD44 ＋/CD24-比率为23.77％,而LACC肿瘤微球体CD44 ＋/CD24-比率为84.25％.在含血清环境下LACC肿瘤细胞微球体可贴壁分化为普通的腺样囊性癌细胞.结论 用含有生长因子的SFM悬浮人泪腺腺样囊性癌细胞系LACC可获得泪腺腺样囊性癌肿瘤细胞微球体,此肿瘤微球体中富集肿瘤干细胞,为人泪腺腺样囊性癌肿瘤干细胞的研究提供了一个理想的模型系统.

Objective To find ways to isolate and culture lacrimal adenoid cystic carcinoma （LACC） stem cells from human LACC cell line and to identify their biological properties.Methods Experimental research.LACC cells were digested,centrifuged and suspended in specific serum free medium （SFM） to get LACC cancer stem cell spheres.Limiting dilution analysis and monoclonal formation assay were performed to determine the proportion of cancer stem cells in LACC cell line.MTT assay was used to determine the proliferation and chemotherapeutic drug resistance of stem cell spheres.The expressions of cell surface markers CD44 ＋/CD24-were detected by flow cytometry.Stem cell spheres differentiation were induced by dropping serum medium into SFM and the morphologic changes were observed.The IC5o and UV optical density of two kinds of cells were tested by Student＇s t-test.Results About （0.92 ± 0.02） % cells were clonogenic in LACC cell line.They could survive and proliferate to form free-floating cell spheres in SFM,which could stably passaged.The IC5o values of cancer stem cells and LACC cell line were 22.53 mg/L and 11.06 mg/L （t =37.94,P 〈 0.05）,which suggested that cancer stem cells were more resistant to cisplatinum than LACC cell line.In flow cytometry assay,84.25% stem cell spheres were CD44 ＋/CD24-cells,comparing with 23.77% in LACC cell line.Stem cell spheres could differentiate into normal adenoid cystic carcinoma cells in medium with serum.Conclusion LACC stem cell spheres,containing a large number of cancer stem cells,could be obtained by serum free suspension culture.This provide us an ideal model system for cancer stem cell research.