基于3D细胞培养系统的肺癌类干细胞的分离和鉴定

Isolation and identification of lung cancer stem-like cells based on a 3D cell culture system

目的:本研究建立三维细胞培养方法(3D)以分离和鉴定肺癌类干细胞,并与二维细胞培养方法(2D)比较,初步探讨该方法在评价肺癌体外增殖、凋亡、侵袭和药物反应性方面的应用。方法:将人肺腺癌细胞系A549以1×104个/孔的细胞与RPMI1640和伊格尔基础培养基(basal medium of eagle,BME)制成细胞悬液,培养7 d,收集细胞采用流式细胞仪进行类干细胞表型检测,以及体外成球、耐药性、体内成瘤等干细胞功能鉴定,并将3D与2D细胞培养的A549细胞进行比较。结果:细胞表型检测证明3D系统培养的A549细胞中CD44和CD326阳性比例升高,CD24阳性比例下降,细胞体外成球率显著升高(28.50%±1.17%vs.8.67%±0.80%,P<0.01)对顺铂耐药性显著提高。加药后48 h为58.17%±2.19%vs.33.27%±5.76%(P<0.01),加药后72 h为41.70%±5.81%vs.27.30%±4.25%(P<0.01),体内成瘤时间显著缩短[(20.75±0.85)d vs.(60.25±1.49)d,P<0.01]。结论:3D培养的肺癌细胞中获得更高比例的类干细胞样细胞,具有体内成瘤快、体外成克隆团率高、耐药性提高的特征。

Objective：To highlight the developmental process of 3D cell culture technology system, which is more suitable for isolating and identifying lung cancer stem-like cells than 2D cell culture technology system, and to explore the application of 3D cell cultures in the evaluation of proliferation, apoptosis, invasion, and drug resistance of lung cancer. Methods：Cells （104/well） from the human lung adenocarcinoma cell lines A549 and RPMI 1640 were cultured in complete medium containing 10%fetal bovine serum. Cell suspension was cultured in a BME basal medium. A growth curve was drawn after 7 d of culture. The stem-like cell was identified through a mammosphere culture, drug resistance and invasion assay, and flow cytometry. Data of A549 cells cultured in 3D and 2D tra-ditional cell culture technologies were compared. Results： Cells from the 3D cell culture had higher tumor formation rates [（20.75 ± 0.85） d vs. （60.25 ± 1.49） d, P〈0.01）] and tumor sphere formation （28.50%± 1.17%vs. 8.67%± 0.80%, P〈0.01） than those from the 2D cell culture. Moreover, cells from 3D cell culture were more invasive and resistant to therapy （58.17%± 2.19%vs. 41.70%±5.81%in 48 h, P〈0.01;33.27%±5.76%vs. 27.30%±4.25%in 72 h, P〈0.01）. Phenotype experimental results demonstrated that the CD44 and CD326 cells were double-positive, whereas the CD24 cell was negative. Conclusion：The proportion of stem-like cells in A549 cell line after 3D cell culture significantly increased compared with 2D cell culture. The 3D cell culture can promote the proliferation of lung cancer stem cells.