摘要
目的以食管癌细胞株Eca109为实验材料,采用无血清法培养出球样细胞,在体外大量繁殖,运用多种方法鉴定其干细胞特性,为研究食管癌创造条件。方法在含10%小牛血清的细胞培养液中培养食管癌细胞株Eca109,传2～3代,后逐步降低细胞培养液中小牛血清的浓度,直至无血清,1周后收集悬浮生长的球样细胞,采用流式细胞仪分析细胞周期;免疫荧光法检测干细胞标记物亮氨酸丰富重复G蛋白偶联受体5重组蛋白(Lgr5)的表达;裸鼠致瘤实验检验球样细胞体内成瘤情况。结果 Eca109细胞在无血清培养液中培养1周,在上清液中收集少量悬浮生长的球样细胞,进行消化、传代,得到大量悬浮生长的球样细胞。比较Eca109细胞和球样细胞的细胞周期,Eca109细胞有(28.89±6.34)%处于分裂期,(52.12±4.53)%处于分裂前期;球样细胞有(6.48±2.35)%处于分裂期,(77.19±4.52)%处于分裂前期,两者比较差异有统计学意义(P<0.05)。Eca109细胞Lgr5表达率为(22.67±3.62),而球样细胞中Lgr5的表达率均为100%,两者比较差异有统计学意义(P<0.05)。裸小鼠成瘤实验发现,Eca109细胞无法在裸小鼠皮下成瘤,球样细胞只需2×10~4个即能成瘤,两者比较差异有统计学意义(P<0.05),且随着接种数目增多,瘤体体积也增大。结论逐步降低细胞培养液中小牛血清的浓度直至无血清条件,可驯化人食管癌细胞株Eca109繁殖出球样细胞,表现出肿瘤干细胞特性。
Objective To generate Suspended spheres, Esophageal cancer cell line Eca109 cells were acclimated gradually in the free serum culture medium, 1 week later, a great quantity of spheres were obtained for the further research. Methods Eca109 cells and spheres were collected and stained by PI, Cell cycle distribution was then acquired by flow cytometry. Eca109 cells and spheres were incubated with anti-Lgr5 Ab and then detected with confocal microscopy. Eca109 cells and spheres were injected subcutaneously into the lateral root of two posterior limb of a nude mouse, tumor volume was calculated. Results Spheres formed when grown under serum-free conditions in 7 days, and the ability of Eca109 cells to form spheres was sustained. It had been shown that the spheres exhibited a relatively high proportion of cells in G0/G1 phase(77.19±4.52)% and low proportion in S phase(6.48±2.35)%;when compared to Eca109 cells,G0/G1 phase(52.12±4.53)%, S phase(28.89±6.34)%(P<0.05). A few of Eca109 cells be cultured in DF+10% FBS clearly showed the expression of pluripotent stem cell markers Lgr5(22.67±3.62)%, but almost all spheres expressed the marker and there was a significant difference between the two groups in the expression ratio(P<0.01). 2×10^4 sphere cells injected subcutaneously into the lateral root of one posterior limb of a nude mouse formed transplantation tumor after 4 weeks, and the volume of transplantation tumor was increased with the cell number increased;Eca109 cells could not form transplantation tumor(P<0.05). Conclusion In this study, we observed successfully that Eca109 cells being cultured in serum-free medium resulted in spheres rapidly and the spheres were serially passaged with relatively high efficiency. The spheres showed the characteristics of the cancer stem cell.
作者
杨婷
马磊
蔡少青
梁宏开
YANG Ting;MA Lei;CAI Shaoqing;LIANG Hongkai(Department of Intensive Care Unit, Zhongshan People′s Hospital, Zhongshan Guangdong 528403, China;Department of General Surgery, The Eighth Hospital of Nanhai Region, Foshan Guangdong 5282161, China)
出处
《新疆医科大学学报》
CAS
2019年第4期467-471,共5页
Journal of Xinjiang Medical University
基金
2015年中山市科技计划项目(2015B1090)
2015年广东省医学科研基金(A2015516)
关键词
食管癌
无血清培养
肿瘤干细胞
esophageal cancer
cancer stem cells
serum-free culture
