神经生长因子受体阳性的人食管鳞癌细胞的分选及其干细胞特性

Isolation of nerve growth factor receptor positive cells from human esophageal squamous carcinoma cells and their biological properties

目的应用免疫磁珠分选(magnetic activated cell sorting,MACS)法分选人食管鳞癌细胞(esophagealsquamous carcinoma cell,ESCC)中的神经生长因子受体(nerve growth factor receptor,NGFR)阳性细胞,并对其肿瘤干细胞样特征进行研究。方法采用MACS分选人ESCC中的NGFR阳性细胞。通过流式细胞仪法测定分选细胞的纯度及细胞周期分布,并计算细胞回收率;MTT法测定细胞数,绘制生长曲线并计算细胞群体倍增时间;平板克隆实验测定平板克隆形成率;裸鼠接种成瘤实验测定体内致瘤性,从而鉴定NGFR阳性ESCC的干细胞特性。结果 NGFR阳性细胞约占肿瘤细胞的1.89%,经过磁珠分选后纯度显著提高,达89.94%,细胞回收率为19.83%。与未分选细胞相比,分选后NGFR阳性细胞的增殖速度明显提高,生长曲线左移,细胞倍增时间缩短[(25.22±8.15)hvs.(47.41±12.69)h,P=0.01],平板克隆形成率显著增加[(16.27±8.31)%vs.(1.22±0.35)%,P=0.00],处于活跃增殖期S、G2、M期的细胞显著增加(72.7%vs.18.8%);裸鼠皮下接种的转移瘤形成率增加(8/10vs.1/10),转移瘤重量显著增加[(0.72±0.53)gvs.(0.16±0.26)g,P=0.00]。结论应用MACS技术能有效分选出人ESCC中的NGFR阳性细胞,其具有肿瘤干细胞特性。

Objective To isolate nerve growth factor receptor（NGFR） positive cells from human esophageal squamous carcinoma cells（ESCC） and to observe their biological properties.Methods NGFR positive cells were isolated from human ESCCs by magnetic activated cell sorting（MACS）.NGFR positive rate of isolated cells and cell generation cycles were detected by flow cytometry,meanwhile the cell recovery rate was calculated.Cell numbers were counted by MTT method,cell growth curves were obtained,and doubling generation times were calculated.Flat plate clone test was performed to calculate cloningefficiency rate.Tumor development test was performed by the xenotransplantation of NGFR positive squamous carcinoma cells in Balb/C mice.Results The purity of NGFR positive cells in isolated cells was up to 89.94%（vs.1.89% in non-isolated cells）,while cell recovery rate was 19.83%.Compared with non-isolated cells,NGFR positive cells showed stronger ability of self-renew,differentiation,proliferation,shortening of doubling generation times [（25.22±8.15）h vs.（47.41±12.69）h,P=0.01] and increasing of cloning efficiency rate [（16.27±8.31）% vs.（1.22±0.35）%,P=0.00].Cell generation cycles test indicated that most of cells were in S,G2 and M phase（72.7% vs.18.8%）.Tumor development in Balb/C mice showed larger size and heavier weight in NGFR positive isolated cells in Balb/C mice compared with non-isolated cells [（0.72±0.53）g vs.（0.16±0.26）g,P=0.00].Conclusions NGFR positive cells can be isolated from human ESCCs by MACS with high purity and maintenance of cell biological functions.NGFR positive cells in human esophageal squamous carcinoma shows the properties of cancer stem cells.