无血清悬浮法筛选人结肠癌干细胞样亚群的初步研究

摘要

目的 应用无血清培养基( SFM)悬浮法培养人结肠癌细胞,筛选和鉴定人结肠癌细胞中肿瘤干细胞相关亚群,并对其进行干预.方法 取新鲜的结肠癌肿瘤组织进行原代培养,分离获得的原代结肠癌细胞在SFM中形成肿瘤干细胞球,而在含血清培养基( SSM)中则为贴壁生长.用5-氟尿嘧啶(5 - Fu)对筛选获得的肿瘤干细胞球进行干预,检测侧群细胞(SP)比例、细胞克隆形成能力、正常肠道干细胞标记物Musashi -1表达及结肠癌干细胞特异性标记物CD133的表达.结果 原代培养获得的人结肠癌细胞能够在SFM中存活、增殖并形成肿瘤干细胞球,肿瘤干细胞球的SP细胞含量高、克隆形成率高,且Mushashi -1及CD133的表达均较高,5- Fu对其抑制率较低.经5- Fu干预后,结肠癌肿瘤干细胞球中SP细胞比例增加、Musashi -1及CD133表达增加,克隆形成率无明显差异.结论 SFM的培养法可从人结肠癌细胞中分离获得极少量肿瘤干细胞球,5-Fu干预可再次富集该细胞亚群中的肿瘤干细胞.%Objective To explore the application of serum - free medium (SFM) suspension to culture human colon cancer cells, screen and identify human colon cancer cells in tumor stem cells subsets, and give relevant intervention. Methods Fresh colon tumor tissues were taken and put in pri mary culture. They were then isolated from the primary colon cancer cells to form tumor stem cell ball in SFM and grow adherently in serum - free medium ( SSM). 5 - Fu was used to intervene cancer stem cells ball which were obtained on the screening. Ratio of side population cells (SP) , ability of cell colony formation, expression of normal intestinal stem cell marker Musashi - 1 and expression of colon cancer stem cell - specific marker CD]33 were examined. Results Human colon cancer cells obtained from primary culture could survive, proliferate and form cancer stem cell ball in SFM. SP cells in cancer stem cells ball were high, and they had high rate of clone formation. Mushashi - 1 and CD133 expression were higher, and the inhibition 5 - Fu on SP cells was low. After 5 - Fu intervention, the proportion of SP cells in the colon cancer stem cells ball, the expressions of Musashi - 1 and CD133 in creased. There was no significant difference in cloning formation. Conclusion A very small amount of cancer stem cells ball can be isolated from human colon cancer cells by SFM training method, and 5 - Fu intervention can re - enriched cell subsets in the tumor stem cells.