人鼻咽癌细胞株中类肿瘤干细胞的分离、培养及鉴定

Isolation and identification of tumor-like stem cells from human nasopharyngeal carcinoma cell line

目的检测CD133在人鼻咽癌细胞株SUNE中的表达以及CD133+肿瘤细胞的体外增殖、分化情况,并对其进行类肿瘤干细胞的鉴定。方法采用免疫荧光细胞化学技术及流式细胞仪检测鼻咽癌细胞株SUNE中CD133的表达情况以及CD133+细胞体外分化能力;免疫磁珠分选技术纯化CD133+肿瘤细胞;MTT法检测CD133+细胞的体外增殖能力,并将其与CD133-及未分选细胞进行比较。结果鼻咽癌细胞株SUNE中有0.35%的微量细胞呈CD133+表达;免疫磁珠富集的CD133+肿瘤细胞在无血清培养基中悬浮生长,并可以形成肿瘤细胞球,具有很强的自我更新和繁殖能力,且在3、5、7 d的紫外吸光度(A)值分别为0.317±0.007、0.370±0.002、0.558±0.004,均高于相同条件下未分选细胞和CD133-细胞(P<0.05);CD133+细胞在含血清培养基中的比例逐日下降,至培养的第12天,由第1天的89.67%下降至0.37%。结论CD133可作为鼻咽癌类干细胞的标志之一。鼻咽癌细胞株SUNE中存在类肿瘤干细胞,并具有自我更新和分化能力。

Objective To investigate the expression of CD133 in human nasopharyngeal carcinoma cell line SUNE, and to detect proliferative and differential ability of CD133^＋ tumor cells in vitro, and to identify tumor-like stem cells in SUNE. Methods Immunocyto-chemical staining method and flow cytometry were used to detect the expression of putative tumor-initiated cell marker CD133 in nasopha-ryngeal carcinoma cell line SUNE, and the isolation technique with immunomagnetic beads was applied to purify CD133^＋ cells. The prolif-erative ability of CD133^＋ tumor cells in vitro was estimated by MTT assay, and the proliferative ability of CD133^＋ , CD133 and control SUNE cells were statistically compared. Finally, the immunocytochemical staining method and flow cytometry were used to detect differential ability of CD133^＋ tumor cells in vitro. Results CD133 was positively expressed only in 0.35% of the cells in nasopharyngeal carcinoma cell line SUNE. In the serum-free medium RPM 11640, the UV optical density of the CD133^＋ tumor cells enriched with immunomagnetic beads was 0.317±0.007, 0. 370±0.002 and 0.558±0.004 at 3, 5 and 7 days, respectively. Compared with CD133 cells and control SUNE cells, CD133^＋ cells showed increased proliferative capacity （P〈0.05）. The proportion of CD133^＋ cells declined in the culture medium containing serum with the passage of time. During the period of 12 days of culturing, the CD133^＋ cells decreased gradually from 89.67% to 0.37%. Conclusions CD133 may be assumed as one of markers of tumor-like stem cells existing in human nasopharyngeal carcinoma cell line SUNE, and nasopharyngeal carcinoma-like stem cells may exist in human nasopharyngeal carcinoma cell line SUNE, pos sessing the ability of self-renovation and multidirectional differentiation.