一种大鼠肝癌干细胞的离心富集方法  

A way to enrich the rat hepatic cancer stem cells

作　　者：王兴[1] 刘卫辉[1] 夏宁[1] 张娟 杨建栋[1] 王涛[1] 尤楠[1] 龚振斌[1] 季茹[1] 赵戈[1] 窦科峰[1] 

机构地区：[1]第四军医大学附属西京医院肝胆胰脾外科,西安710032 [2]解放军第四十四医院五官科

出　　处：《中华实验外科杂志》2012年第5期857-860,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81030010、81170419）

摘　　要：目的建立一种富集大鼠肝癌干细胞的方法，并检测此种方法分离得到的细胞是否具有肿瘤干细胞的生物学特征。方法二乙基亚硝胺（DENA）腹腔注射构建大鼠肝癌模型，两步胶原酶灌注法分离肝癌细胞并体外培养，Percoll连续梯度密度离心法将细胞分成4层，采用流式细胞仪检测各层细胞的肝癌干细胞表面标志上皮细胞黏附因子（EpCAM）、甲胎蛋白（AFP）的表达，并用实时一聚合酶链反应（ReM—timePCR）验证EpCAM、CDl33、AFP的mRNA表达。绘制生长曲线检测各层细胞增殖能力，Transwell小室检测各层细胞的侵袭能力。结果Percoll离心所得4层细胞中，第3层细胞是富集肝癌干细胞最多的一层，具有最强的增殖能力。第3层细胞EpCAM与AFP阳性表达最高，分别为（84．50±8．31）％和（85．20±8．31）％，与其他各层比较，差异有统计学意义（P〈0．05）。经ReM—timePCR验证，第3层细胞的EpCAM和AFPmRNA表达最高，分别为（111．34±5．59）和（13．76±0．95），与其他各层比较，差异有统计学意义（P〈0．05），第3层的CD133表达为（17．16±0．42），与第2层、4层比较，差异有统计学意义（P〈0．05），但与第1层比较，差异无统计学意义（P〉0．05）。侵袭实验可见第3层细胞穿透的数量最多（92．750±4．432），与其他各层比较，差异有统计学意义（P〈0．05）。结论利用Percoll连续梯度密度离心法成功地富集了肝癌干细胞，经验证符合肿瘤干细胞生物学特性，为肝癌的靶向治疗、多重耐药等基础研究提供了确切的研究对象。Objective - To establish a novel approach of enriching rat hepatic cancer stem cells （HCSCs） in vitro and identify the biological features of HCSCs. Methods Based on the previously estab- lished three-step method for stem cells isolation, HCSCs were enriched briefly as follows. Firstly the rat liv- er cancer model was made by injecting diethylnitrosamine （ DENA ） intraperitoneally. By using modified Selgen＇s two-step method, the rat liver cancer cells were isolated. After these ceils were cultured for 10 days, the first stet~ was done to purify the ceils with differential trypsinization and differential adherence. When the cells spread more than 90% pavement of culture flask, the second step was done to fraetionate the cells with Percoll continuous gradient centrifugation. To identify the properties of stem cells from each fraction, the third step was done, included detection of three markers of HCSCs [ epithelial cell adhesion molecule （EpCAM）, CD133, alpha fetal protein （AFP）] by using flow cytometry and reverse transcrip- tion-polymerase chain reaction （RT-PCR）, and assay of the multiplication capacity and invasion of cells from every fraction. Results The hepatic cancer cells at the fraction 3 were most likely enriched with HCSCs, which had the highest proliferative ability. The matrigel invasion assay indicated that the largest number （92. 750±4. 432） of cells at the fraction 3 migrated through the membrane, which was significantly different from the others. Flow cytometry revealed that the expression of EpCAM （ 84. 50±8. 31 ） % and AFP （85.20±8. 31 ）% at fraction 3 was significantly different from the others. RT-PCR revealed that the ex- pression of EpCAM （ 111.34 ±5.59） and AFP （ 13.76±0. 95） at the fraction 3 was significantly different from the others. The expression of CD133 at the fraction 3 （ 17.16±0.42） was significantly different from that at the fraction 2 or fraction 4, but insignificantly different from that at the fraction 1. Conclusion A simpl

关 键 词：癌 肝细胞 干细胞 细胞分离 

分 类 号：R735.7[医药卫生—肿瘤]