摘要目的 进行人食管肿瘤干细胞(CSCs)的分选及鉴定.方法 通过获取人食管癌标本进行肿瘤细胞的培养,应用流式细胞仪检测p75NTR在人食管肿瘤细胞(ECCs)中的表达,应用磁珠分选法(MACS)进行p75NTR阳性细胞与阴性细胞的分选,研究p75NTR阳性细胞的增殖、分化、软琼脂集落形成能力,并进行裸鼠接种以观察其致瘤能力.结果 在8个食管肿瘤细胞系(株)中,除SHEC-1、SHEC-5未检测到p75NTR阳性细胞外,其余6个细胞系(株)中检测到p75NTR阳性细胞为0.32%～3.35%.MACS分选后的p75NTR阳性细胞的增殖能力明显强于阴性细胞[群体倍增时间分别为(17±3)h、(37±7)h,P<0.01];p75NTR阳性细胞具有分化产生其他表型细胞的能力;p75NTR阳性细胞在软琼脂中的集落形成能力(45.9%±8.9%)显著高于阴性细胞(3.7%±2.1%),P<0.01;行裸鼠接种时,p75NTR阳性细胞表现出较强的致瘤性,SHEC-7细胞只需2×103个即可致瘤,其致瘤能力是未分选细胞的50倍.结论 人ECCs中p75NTR阳性细胞具有自我更新、分化、增殖能力,并具有较强的致瘤能力,p75NTR阳性细胞具有CSCs的特性.

abstractsObjective To isolate and identify cancer stem cells from esophageal carcinoma cells (ECCs) using cell surface marker p75NTR. Methods ECCs cell lines were established with ECCs collected from 38 surgically resected specimens. Flow cytometry was used to identify the p75NTR positive cells therein that were isolated then using magnetic activated cell sorting (MACS) method. The growing characteristics in DMEM medium and capability of colony-forming in soft agar of the p75NTR positive cells were evaluated ex vivo with MTF method, p75NTR positive cells of different concentrations were subcutaneously injected into the backs of Balb/c nude mice and PBS was injected into the contralateral back, and then tumorigenisis was observed, 8 weeks later the mice were killed with their tumors taken out to undergo microscopy. Results Eight ECCs cell lines were established, 6 of which were found to contain 0. 32% - 3.35% of p75NTR positive cells. The purity of p75NTR positive cells isolated by MACS was up to 90%. MTr result showed that population doubling time of the p75NTR positive cells was (17±3) hours, significantly shorter than that of the p75NTR negative cells [ (37±7) hours, P<0.01 ]. The colony-forming rate in soft agar of the p75NTR positive cells was (45.9%±8.9% ), significantly higher than that of the p75NTR negative cells [ (3.7%± 2.1% ), P<0.01]. As few as 2000 p75NTR positive cells gave rise to new tumors in xenotransplantation, with a tumorigenic ability 50 times as high as that of the p75NTR negative cells. Conclusion p75NTR positive cells carry some properties of cancer stem cells, such as the ability of self-renewal, differentiation and proliferation and demonstrate higher ability of colony-forming ex vivo and tumorigenisis in vivo.