人眼脉络膜黑色素瘤OCM-1细胞中肿瘤干细胞的分离纯化

Isolation and purification of melanoma stem cells in human choroidal melanoma OCM-1 cells

目的分离及纯化人眼脉络膜黑色素瘤细胞OCM-1中的肿瘤干细胞。方法将OCM-1细胞复苏，使用Dubecco标准改良Eagle培养基（DMEM/F12）培养后，改用无血清化学限定培养基（SFM），并分离纯化培养肿瘤干细胞，用流式细胞仪检测神经干细胞特异性标记物CD133的表达比率，并对第六代细胞进行神经RNA结合蛋白（musashi-1）免疫细胞化学染色，显微镜下观察阳性细胞比率。结果在标准DMEM/F12培养基中贴壁生长的OCM-1细胞用SFM培养基培养后，贴壁细胞显著减少，至第6代时细胞均为悬浮团状集落生长，呈现典型的干细胞生长方式。在不同代的细胞中，CD133均有阳性，未经过分离的OCM-1细胞、经过SFM培养并分离的第1、2代OCM-1细胞的阳性比率分别是2.5%、21.7%、57.8%，第6代的细胞CD133阳性比率达到79.8%，musashi-1强阳性表达。结论脉络膜黑色素瘤细胞中存在肿瘤干细胞。使用干细胞培养液培养，并通过限制分化、连续传代的方法，可分离纯化出悬浮克隆集落生长的干细胞。

Objective To isolate and purify the melanoma stem cells （MSC） in choroidal melanoma OCM-1 cells. Methods OCM-1 cells were resuscitated, and after cultured in standard Dubecco＇s modifided Eagle＇s medium （DMEM）/F12, they were cultured in serum-free medium （SFM）. The cultured MSC were isolated and purified, and the positive rate of CD133, the specific markers of neuro-stem cells, was observed by flow cytometry （FCM）. The 6th generation of the cells were stained by musashi-1 immunocytochemistry, and the rate of the positive cells was observed under the microscope. Results After the Adherent OCM-1 cells cultured in SFM, the number of the adherent number decreased obviously. The cells at the 6th generation grew as the suspended gobbets, which represented the typical grow manner of the stem cells. Positive CD133 could be found in the cells of different generations, which was 2.5%, 21.7%, and 57.8% in the non-isolated OCM-1 cells, the 1st generation of isolated cells, and the 2nd generation cells, respectively. The positive rate of CD133 in the cells at the sixth generation was 79.8% with strong positive expression of musashi-1. Conclusion MSC is in the human choroidal melanoma OCM-1 cells. The suspended stem cells may be purified by limited differentiation and serial passage in SFM.