5-氟尿嘧啶对肝癌细胞系BEL-7402中肿瘤干细胞的富集作用  

The effect of 5 - fluorouracil on enriching cancer stem cells of hepatoma cell line BEL-7402.

作　　者：杨玥[1,3] 李德龙[3] 朱雯静[3] 刘菲[3] 康梦田[3] 赵森[3] 郝溥辰[3] 韩旭[3] 陈培琼[3] 吕福东[1] 冯骥良[1,2] 

机构地区：[1]首都医科大学附属北京佑安医院病理科,北京100069 [2]首都医科大学基础医院病理教研室 [3]首都医科大学七年制研究生

出　　处：《中华肝脏病杂志》2011年第9期686-691,共6页Chinese Journal of Hepatology

基　　金：北京市教育委员会“人才强教”项目中青年骨干人才培养计划基金（PHR201008387）

摘　　要：目的探讨5-氟尿嘧啶（5-FU）对肝癌细胞系BEL-7402细胞的作用，并对免于5-FU杀伤的存活细胞进行干细胞表面标志物的检测和“肝癌干细胞”的细胞生物学鉴定。方法检测5-FU作用前后BEL-7402细胞增殖能力、细胞周期和细胞表面标志物CD56、CD54、上皮细胞黏附分子（EpCAM）和CD133表达率的变化；通过集落形成实验检测CD56、CD54、EpCAM、CD133阳性细胞和阴性细胞的克隆形成能力并对其进行比较。两组间均数比较采用t检验，两组数据间构成比比较采用X^2检验。结果5-FU对BEL-7402细胞的增殖抑制作用呈时间和剂量依赖性。10ug／ml5-Fu作用48h后，G0／G1期细胞比例由对照组的57．50％±0．98％升高至实验组的68．70％±3．41％（P〈0．05）;S期细胞比例由对照组的40．26％±4．12％下降至实验组的31．80％±4．15％（P〈0．01）；实验组G2／M期细胞消失，对照组该期细胞比例为5．80％±1．87％（P〈0．01）。细胞周期分布变化明显，差异具有统计学意义。5-Fu处理使BEL-7402细胞中CD56、CD54、EpCAM和CD133阳性细胞比例分别由0．57％±0．12％，8．10％±6．79％，0．3％±0．010／0，3．20%±0．99％升高至4．13%±0．06％，50．08％±1．69％，0．55％±0．07％，10．51％±1．13％，差异具有统计学意义（P〈0．05）。软琼脂集落形成实验中，CD56、CD54、EpCAM、CD133阴性细胞与阳性细胞的成集落比例分别为2．11％±0．21％，3．32％±0．310／0；0．86％±0．101％，2．40±0．52%;7．19％±0．56％，7．73±0．71％；2．70％±0．26％，5．75％±0．81％。各组阳性细胞集落形成率与阴性细胞比较，均有明显增加，差异具有统计学意义（P〈0．05）。结论-定剂量的5-FU能够富集肝癌细胞系BEL-7402中的肿瘤干细胞；同EpCAM和CD133一样，CD56和CD54单阳性细胞比其各自单阴性细胞具有更强的克隆形成能力，这提示CD56和CD54可�Objective To investigate the effect of 5-FU （5 - fluorouracil） on enriching cancer stem cells of HCC cell line BEL-7402 and the biological characteristics of enriched cells. Methods The enriching concentration of 5-FU was determined by CCK-8 （cell counting kit-8）. Flow Cytometry was used to determine the changes in cell cycle and positive expression ratio of surface marker CD56, CD54, EpCAM and CD133.The self-renewal and differentiation of positive ceils were tested by colony formation assay, and were compared with the control group. Results Enriching concentration of 5-FU was determined as 10~tg/ml with 48h incubation. After enrichment, G0/G1 phase cells increased from 57.50 %±0.98% to 68.70%±3.41% （P 〈0.05）. Whereas S phase cells decreased from 40.26%±4.12% to 31.80%±4.15% （P 〈0.01）; G2/M phase cells disappeared in experimental group, and was 5.80%± 1.87% in control group （P 〈0.01）. The proportion of the cell cycle changed with significant statistical differences. Meanwhile, positive rate of cell surface makers CD56, CD54, EpCAM and CD133 increased from 0.57% ± 0.12%, 8.10% ± 6.79%, 0.3% ± 0.01% and 3.20% ±0.99% to 4.13% ± 0.06%, 50.08% ± 1.69%, 0.55%± 0.07% and 10.51% ± 1.13%, respectively. The difference was significant （P 〈 0.05）. The colony forming ratio of CD56, CD54, EpCAM and CD133 negative cells and positive cells were 2.11% ± 0.21%, 3.32% ＋ 0.31%; 0.86% ± 0.101%, 2.40% ± 0.52 %; 7.19%± 0.56%, 7.73% ± 0.71%; 2.70% ± 0.26%, 5.75% ± 0.81%, respectively, and significant differences were found between （P 〈0.05）. Conclusion 5- fluorouracil enriched the cancer stem cell population in HCC cell line BEL-7402. CD56 and CD54 can be used as important surface markers in research of liver cancer stem cells.

关 键 词：癌 肝细胞 肿瘤干细胞 氟尿嘧啶 CD56 CD54 

分 类 号：R735.7[医药卫生—肿瘤]